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Histone deacetylase inhibition protects hearing against acute ototoxicity by activating the Nf-κB pathway.

Layman WS, Williams DM, Dearman JA, Sauceda MA, Zuo J - Cell Death Discov (2015)

Bottom Line: We also found that the apparent lack of hair cell loss was completely independent of spontaneous or facilitated (ectopic Atoh1 induction) hair cell regeneration.In addition, we also detected increased expression of pro-survival genes Cdkn1a (p21) and Hspa1a (Hsp70), and decreased expression of the pro-apoptosis gene Bcl2l11 (Bim).These data combined provide evidence that class I HDACs control the transcriptional activation of pro-survival pathways in response to ototoxic insult by regulating the acetylation status of transcription factors found at the crossroads of cell death and survival in the mammalian inner ear.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Developmental Neurobiology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.

ABSTRACT

Auditory hair cells have repeatedly been shown to be susceptible to ototoxicity from a multitude of drugs including aminoglycoside antibiotics. Here, we found that systemic HDAC inhibition using suberoylanilide hydroxamic acid (SAHA) on adult mice offers almost complete protection against hair cell loss and hearing threshold shifts from acute ototoxic insult from kanamycin potentiated with furosemide. We also found that the apparent lack of hair cell loss was completely independent of spontaneous or facilitated (ectopic Atoh1 induction) hair cell regeneration. Rather, SAHA treatment correlated with RelA acetylation (K310) and subsequent activation of the Nf-κB pro-survival pathway leading to expression of pro-survival genes such as Cflar (cFLIP) and Bcl2l1 (Bcl-xL). In addition, we also detected increased expression of pro-survival genes Cdkn1a (p21) and Hspa1a (Hsp70), and decreased expression of the pro-apoptosis gene Bcl2l11 (Bim). These data combined provide evidence that class I HDACs control the transcriptional activation of pro-survival pathways in response to ototoxic insult by regulating the acetylation status of transcription factors found at the crossroads of cell death and survival in the mammalian inner ear.

No MeSH data available.


Related in: MedlinePlus

Hair cell regeneration is not facilitated by SAHA. Fgfr3-icreERT2; Atoh1-HA; tdTomato mice treated with SAHA with or without kanamycin did not generate new hair cells. (a and b) Fgfr3-icreERT2; Atoh1-HA; tdTomato mice treated with vehicle (0.9% saline) had normal-appearing outer hair cells that lack the formation of newly generated hair cells regardless of whether SAHA or vehicle (DMSO) was delivered. (c and d) Kanamycin administered to Fgfr3-icreERT2; Atoh1-HA; tdTomato mice that were treated with vehicle (DMSO) lost the vast majority of their outer hair cells, whereas SAHA-treated kanamycin mice retained their outer hair cells and did not form new hair cells. All the representative images were taken from the middle turn of the cochlea. Scale bar is 20 μm.
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Figure 4: Hair cell regeneration is not facilitated by SAHA. Fgfr3-icreERT2; Atoh1-HA; tdTomato mice treated with SAHA with or without kanamycin did not generate new hair cells. (a and b) Fgfr3-icreERT2; Atoh1-HA; tdTomato mice treated with vehicle (0.9% saline) had normal-appearing outer hair cells that lack the formation of newly generated hair cells regardless of whether SAHA or vehicle (DMSO) was delivered. (c and d) Kanamycin administered to Fgfr3-icreERT2; Atoh1-HA; tdTomato mice that were treated with vehicle (DMSO) lost the vast majority of their outer hair cells, whereas SAHA-treated kanamycin mice retained their outer hair cells and did not form new hair cells. All the representative images were taken from the middle turn of the cochlea. Scale bar is 20 μm.

Mentions: Studies have shown that inhibitors of epigenetic events, such as DNA methylation, histone deacetylation, and histone methylation, are able to facilitate cellular reprogramming.13–18 Although unlikely, it is possible that SAHA may not actually be providing protection against kanamycin ototoxicity but instead SAHA may facilitate hair cell regeneration. To determine whether SAHA is able to induce hair cell regeneration, we analyzed Fgfr3-icreERT2; CAG-loxP-stop-loxP-Atoh1-HA; Rosa26-CAG-loxP-stop-loxP-tdTomato (Fgfr3-icreERT2; Atoh1-HA; tdTomato) mice for hair cell regeneration. In the auditory field, ectopic expression of the transcription factor Atoh1 has been used to convert neonatal mammalian non-sensory cells into cells that express many endogenous hair cell markers.19–21 Although, ectopic expression of Atoh1 alone can convert neonatal non-sensory cells into hair cell-like cells,19–21 loss of cellular plasticity at later postnatal ages prevents this conversion from occurring. At P28, Fgfr3 is not expressed in hair cells but is highly expressed in the non-sensory supporting cells that lie beneath the outer hair cells. As supporting cells are the source of newly regenerated hair cells in non-mammalian vertebrates, the inclusion of the tdTomato reporter in our mouse model allowed us to lineage trace these cells. Intraperitoneal injection of 0.25 mg/g tamoxifen in corn oil was given to Fgfr3-icreERT2; Atoh1-HA; tdTomato mice at P28. Mice were then treated with 100 mg/kg SAHA or vehicle at P30 (one day before acute damage), then at P31 (8 h after kanamycin/furosemide treatment), and at P32. Mice received 600 mg/kg kanamycin or vehicle (0.9% saline) with 400 mg/kg furosemide at P31, euthanized at P44, then processed for immunofluorescence, and analyzed for morphology (n = 6 for each treatment group). We were unable to detect the formation of newly generated hair cells in Fgfr3-icreERT2; Atoh1-HA; tdTomato mice that were treated with SAHA regardless of whether kanamycin was administered (Figures 4a–d). Since ectopic Atoh1 expression in the supporting cells in conjunction with SAHA treatment should have provided the best case scenario for SAHA-mediated regeneration, we concluded that the hair cells found in our wild-type model were protected against ototoxic cell death and not newly regenerated hair cells.


Histone deacetylase inhibition protects hearing against acute ototoxicity by activating the Nf-κB pathway.

Layman WS, Williams DM, Dearman JA, Sauceda MA, Zuo J - Cell Death Discov (2015)

Hair cell regeneration is not facilitated by SAHA. Fgfr3-icreERT2; Atoh1-HA; tdTomato mice treated with SAHA with or without kanamycin did not generate new hair cells. (a and b) Fgfr3-icreERT2; Atoh1-HA; tdTomato mice treated with vehicle (0.9% saline) had normal-appearing outer hair cells that lack the formation of newly generated hair cells regardless of whether SAHA or vehicle (DMSO) was delivered. (c and d) Kanamycin administered to Fgfr3-icreERT2; Atoh1-HA; tdTomato mice that were treated with vehicle (DMSO) lost the vast majority of their outer hair cells, whereas SAHA-treated kanamycin mice retained their outer hair cells and did not form new hair cells. All the representative images were taken from the middle turn of the cochlea. Scale bar is 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Hair cell regeneration is not facilitated by SAHA. Fgfr3-icreERT2; Atoh1-HA; tdTomato mice treated with SAHA with or without kanamycin did not generate new hair cells. (a and b) Fgfr3-icreERT2; Atoh1-HA; tdTomato mice treated with vehicle (0.9% saline) had normal-appearing outer hair cells that lack the formation of newly generated hair cells regardless of whether SAHA or vehicle (DMSO) was delivered. (c and d) Kanamycin administered to Fgfr3-icreERT2; Atoh1-HA; tdTomato mice that were treated with vehicle (DMSO) lost the vast majority of their outer hair cells, whereas SAHA-treated kanamycin mice retained their outer hair cells and did not form new hair cells. All the representative images were taken from the middle turn of the cochlea. Scale bar is 20 μm.
Mentions: Studies have shown that inhibitors of epigenetic events, such as DNA methylation, histone deacetylation, and histone methylation, are able to facilitate cellular reprogramming.13–18 Although unlikely, it is possible that SAHA may not actually be providing protection against kanamycin ototoxicity but instead SAHA may facilitate hair cell regeneration. To determine whether SAHA is able to induce hair cell regeneration, we analyzed Fgfr3-icreERT2; CAG-loxP-stop-loxP-Atoh1-HA; Rosa26-CAG-loxP-stop-loxP-tdTomato (Fgfr3-icreERT2; Atoh1-HA; tdTomato) mice for hair cell regeneration. In the auditory field, ectopic expression of the transcription factor Atoh1 has been used to convert neonatal mammalian non-sensory cells into cells that express many endogenous hair cell markers.19–21 Although, ectopic expression of Atoh1 alone can convert neonatal non-sensory cells into hair cell-like cells,19–21 loss of cellular plasticity at later postnatal ages prevents this conversion from occurring. At P28, Fgfr3 is not expressed in hair cells but is highly expressed in the non-sensory supporting cells that lie beneath the outer hair cells. As supporting cells are the source of newly regenerated hair cells in non-mammalian vertebrates, the inclusion of the tdTomato reporter in our mouse model allowed us to lineage trace these cells. Intraperitoneal injection of 0.25 mg/g tamoxifen in corn oil was given to Fgfr3-icreERT2; Atoh1-HA; tdTomato mice at P28. Mice were then treated with 100 mg/kg SAHA or vehicle at P30 (one day before acute damage), then at P31 (8 h after kanamycin/furosemide treatment), and at P32. Mice received 600 mg/kg kanamycin or vehicle (0.9% saline) with 400 mg/kg furosemide at P31, euthanized at P44, then processed for immunofluorescence, and analyzed for morphology (n = 6 for each treatment group). We were unable to detect the formation of newly generated hair cells in Fgfr3-icreERT2; Atoh1-HA; tdTomato mice that were treated with SAHA regardless of whether kanamycin was administered (Figures 4a–d). Since ectopic Atoh1 expression in the supporting cells in conjunction with SAHA treatment should have provided the best case scenario for SAHA-mediated regeneration, we concluded that the hair cells found in our wild-type model were protected against ototoxic cell death and not newly regenerated hair cells.

Bottom Line: We also found that the apparent lack of hair cell loss was completely independent of spontaneous or facilitated (ectopic Atoh1 induction) hair cell regeneration.In addition, we also detected increased expression of pro-survival genes Cdkn1a (p21) and Hspa1a (Hsp70), and decreased expression of the pro-apoptosis gene Bcl2l11 (Bim).These data combined provide evidence that class I HDACs control the transcriptional activation of pro-survival pathways in response to ototoxic insult by regulating the acetylation status of transcription factors found at the crossroads of cell death and survival in the mammalian inner ear.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Developmental Neurobiology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.

ABSTRACT

Auditory hair cells have repeatedly been shown to be susceptible to ototoxicity from a multitude of drugs including aminoglycoside antibiotics. Here, we found that systemic HDAC inhibition using suberoylanilide hydroxamic acid (SAHA) on adult mice offers almost complete protection against hair cell loss and hearing threshold shifts from acute ototoxic insult from kanamycin potentiated with furosemide. We also found that the apparent lack of hair cell loss was completely independent of spontaneous or facilitated (ectopic Atoh1 induction) hair cell regeneration. Rather, SAHA treatment correlated with RelA acetylation (K310) and subsequent activation of the Nf-κB pro-survival pathway leading to expression of pro-survival genes such as Cflar (cFLIP) and Bcl2l1 (Bcl-xL). In addition, we also detected increased expression of pro-survival genes Cdkn1a (p21) and Hspa1a (Hsp70), and decreased expression of the pro-apoptosis gene Bcl2l11 (Bim). These data combined provide evidence that class I HDACs control the transcriptional activation of pro-survival pathways in response to ototoxic insult by regulating the acetylation status of transcription factors found at the crossroads of cell death and survival in the mammalian inner ear.

No MeSH data available.


Related in: MedlinePlus