c-Myb protects cochlear hair cells from cisplatin-induced damage via the PI3K/Akt signaling pathway

Abstract

The transcription factor c-Myb is vital for cell survival, proliferation, differentiation, and apoptosis. We have previously reported that c-Myb knockdown exacerbates neomycin-induced damage to cochlea cells. However, the function and regulation of c-Myb in the mammalian inner ear remains unclear. Here, we first found that the expression of c-Myb in cochlear HCs was downregulated after cisplatin damage in vivo. Next, to investigate the role of c-Myb in HCs treated with cisplatin, the recombinant virus AAV-ie-CAG-Myb-HA (AAV-c-Myb) that overexpresses c-Myb was constructed and transfected into HCs. The protein expression of c-Myb was effectively up-regulated in cultured cochlear HCs after the virus transfection, which increased cochlear HC viability, decreased HC apoptosis and reduced intracellular reactive oxygen species (ROS) levels after cisplatin injury in vitro. The overexpression of c-Myb in HCs after AAV-c-Myb transfection in vivo also promoted HC survival, improved the hearing function of mice and reduced HC apoptosis after cisplatin injury. Furthermore, c-Myb-HC conditional knockout mice (Prestin; c-Myb-cKO) in which c-Myb expression is downregulated only in cochlear OHCs were generated and the cisplatin-induced HCs loss, apoptosis and hearing deficit were all exacerbated in Prestin; c-Myb-cKO mice treated with cisplatin in vivo. Finally, mechanistic studies showed that upregulation of the PI3K/Akt signaling pathway by c-Myb contributed to the increased HC survival after cisplatin exposure in vitro. The findings from this work suggest that c-Myb might serve as a new target for the prevention of cisplatin-induced HC damage and hearing loss.

Fig. 1. The expression of c-Myb in cochlear HCs is downregulated after cisplatin exposure.

A The ABR thresholds shift of all frequencies were increased after cisplatin administration in mice compared to the control group. B Cell counting and cochleogram there was a significant HCs loss in the cochlear basal turn of the cisplatin group compared to that of the control group, but no significant HCs loss was found in the apical and middle turns in the cisplatin group versus control group. C Representative immunostaining images showing that the fluorescence intensity of c-Myb was significantly downregulated after cisplatin administration compared with the control group. Scale bar = 20 μm. D Western blot showed that the protein level of c-Myb was significantly downregulated in the cisplatin group compared with the control group. n = 6 for each group. *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig. 2. Overexpression of c-Myb increases cochlear HC viability after cisplatin exposure.

A–D Immunofluorescence staining and cell counting showed that cisplatin and AAV-ie co-treatment caused significant IHCs and OHCs loss in all three turns compared to control (AAV-ie) group. The loss of IHCs and OHCs in all three turns were less prominent in cisplatin + AAV-c-Myb group compared with the cisplatin + AAV-ie group. Scale bar = 20 μm.***p < 0.001, n = 6 for each group.

Fig. 3. c-Myb overexpression decreases cochlear HC apoptosis induced by cisplatin.

A Representative images of TUNEL staining showing that apoptotic cochlear HCs exhibited both TUNEL (red) and myosin7a (green) fluorescence after cisplatin treatment. Scale bar (low magnification) = 20 μm. Scale bar (high magnification) = 5 μm. B Statistical analysis verified that the numbers of TUNEL/myosin 7a double-positive IHCs and OHCs in the cisplatin + AAV-c-Myb group were reduced significantly compared to the cisplatin + AAV-ie group. C Representative images of cleaved-caspase3 staining showing that cleaved-caspase3 fluorescence (red) was present in the apoptotic HCs (myosin 7a labeling, green) after cisplatin exposure. Scale bar (low magnification) = 20 μm. Scale bar (high magnification) = 5 μm. D Cell counting and statistical analysis confirmed that the number of cleaved-caspase3/myosin 7a double-positive IHCs and OHCs in the cochlear basal turns was increased significantly after cisplatin treatment compared to the vehicle control group, whereas the number of double-positive cells was decreased significantly in the cisplatin + AAV-c-Myb group compared with the cisplatin + AAV-ie group. **p < 0.01, ***p < 0.001. n = 6 for each group.

Fig. 4. The upregulation of c-Myb expression reduces the level of ROS in cochlear HCs after cisplatin exposure.

A Representative images of Mito-SOX Red staining showing the accumulation of ROS as indicated by the intensified immunostaining signals of Mito-SOX Red (red) in cultured HCs (myosin 7a, green). Scale bar (low magnification) = 20 μm. Scale bar (high magnification) = 5 μm. B A significant number of Mito-SOX/myosin 7a double-positive IHCs and OHCs were observed after cisplatin treatment for 48 h in vitro, while pretreatment with AAV-c-Myb significantly reduced the number of cisplatin-induced Mito-SOX Red/myosin 7a double-positive HCs in the cultured basal-turn cochlear HCs. ***p < 0.001, n = 6 for each group.

Fig. 5. Overexpression of c-Myb in cochlear HCs in vivo improves HC survival and hearing function, protects ribbon synapse in mice after cisplatin injury.

A The ABR threshold shifts at all frequencies from 4 to 32 were significantly reduced in the cisplatin + AAV-c-Myb compared to those in the cisplatin + AAV-ie group. B–E The immunostaining results and cochleogram showed that cochlear HCs pre-treated with AAV-c-Myb reduced the loss of HCs in the basal turns compared to cisplatin + AAV-ie group, but no significant difference of HC loss in the apial and middle turns of cochlea was found in cisplatin + AAV-c-Myb group compared with the cisplatin + AAV-ie group. Scale bar = 20 μm. F, G The average number of Ctbp2 puncta per IHC was significantly reduced in apex, middle and base turns of the cochlea after cisplatin treatment compared to the vehicle control, and the c-Myb overexpression by AAV-c-Myb protected against the loss of Ctbp2 puncta per IHC in all three turns of cochlea after cisplatin administration. Scale bar = 10 μm. ***p < 0.001, n = 6 for each group.

Fig. 6. Overexpression of c-Myb in vivo inhibits cisplatin-induced cochlear HC apoptosis.

A, B Representative images of TUNEL staining and cell counting showing that the numbers of TUNEL/Myosin7a double-positive IHCs and OHCs in the basal turns were increased significantly after cisplatin exposure in vivo compared to the vehicle control group, while the transfection of AAV-c-Myb led to a significantly reduced number of TUNEL/myosin7a double-positive HCs in the cisplatin + AAV-c-Myb group compared to the cisplatin + AAV-ie group. Scale bar = 20 μm. **p < 0.01, ***p < 0.001, n = 6 for each group.

Fig. 7. c-Myb deficiency in OHCs exacerbated cisplatin-induced OHCs loss, apoptosis and hearing deficit in vivo.

A ABR threshold shifts at all frequencies from 4 to 32 kHz were significantly increased in the cisplatin + Prestin; c-Myb-cKO mice compared to those in the cisplatin group. B–E A significant increased OHC loss was found within the Prestin; c-Myb-cKO mice treated with cisplatin compared to the WT mice treated with cisplatin. F, G Basal turns of cochlear HCs were used for apoptosis analysis, and greater number of TUNEL/myosin7a double-positive OHCs were discovered in cisplatin + Prestin; c-Myb-cKO group compared to cisplatin group. Scale bar = 20 μm. **p < 0.01, ***p < 0.001, n = 6 for each group.

Fig. 8. Upregulation of c-Myb reduces the cochlear HC damage induced by cisplatin by activating the PI3K/Akt signaling pathway.

A Western blot showed a significantly decreased ratio of P-Akt/Akt and decreased level of P-PI3K (p85α) in the cultured HCs treated with cisplatin + AAV-ie for 48 h. The levels of P-PI3K (p85α) and P-Akt were significantly reduced in HCs co-treated with cisplatin, AAV-ie and LY294002 compared to the cisplatin + AAV-ie group, while they were significantly increased in HCs co-treated with AAV-c-Myb and cisplatin. The treatment with LY294002 decreased the above effects of c-Myb on p85α level and the ratio of P-Akt/Akt in the cisplatin + AAV-c-Myb + LY294002 group compared to the cisplatin + AAV-c-Myb group. B–F Immunostaining and cell counting showed that the overexpression of c-Myb reduced HC loss after cisplatin injury, but the treatment with LY294002 significantly increased the HC loss in the cisplatin + AAV-c-Myb + LY294002 group compared to the cisplatin + AAV-c-Myb group. Scale bar = 20 μm. ***p < 0.001, n = 6 for each group.