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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

Nf-κB transcription factor RelA retains K310 acetylation resulting in increased expression of pro-survival genes. Taqman gene expression assays were done on microdissected organ of Corti from animals treated with a combination of SAHA/vehicle (DMSO) and kanamycin. Expression levels are relative to endogenous gene controls 18S, GAPDH, and Actb. Fold changes are shown relative to vehicle (DMSO)+kanamycin controls. (a) The expression levels of pro-survival genes Cflar, Bcl2l1, Cdkn1a, and Hspa1a were all increased significantly in SAHA+kanamycin-treated mice but the pro-apoptosis gene Bcl2l11 was significantly decreased compared with vehicle (DMSO) controls. Immunofluorescence of whole-mount organ of Corti was performed using antibodies against RelA/p65 K310 acetylation (red), Prestin (white), Pvalb (green), and Hoechst (blue). (b–e) We found that mice treated with SAHA and kanamycin retained RelA/65 K310 acetyl in the nucleus of the outer hair cells but mice treated with vehicle (DMSO) and kanamycin had a complete absence of RelA/65 K310 acetyl in the nucleus. All the representative images were taken from the middle turn of the cochlea. Scale bar is 20 μm.
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Figure 5: Nf-κB transcription factor RelA retains K310 acetylation resulting in increased expression of pro-survival genes. Taqman gene expression assays were done on microdissected organ of Corti from animals treated with a combination of SAHA/vehicle (DMSO) and kanamycin. Expression levels are relative to endogenous gene controls 18S, GAPDH, and Actb. Fold changes are shown relative to vehicle (DMSO)+kanamycin controls. (a) The expression levels of pro-survival genes Cflar, Bcl2l1, Cdkn1a, and Hspa1a were all increased significantly in SAHA+kanamycin-treated mice but the pro-apoptosis gene Bcl2l11 was significantly decreased compared with vehicle (DMSO) controls. Immunofluorescence of whole-mount organ of Corti was performed using antibodies against RelA/p65 K310 acetylation (red), Prestin (white), Pvalb (green), and Hoechst (blue). (b–e) We found that mice treated with SAHA and kanamycin retained RelA/65 K310 acetyl in the nucleus of the outer hair cells but mice treated with vehicle (DMSO) and kanamycin had a complete absence of RelA/65 K310 acetyl in the nucleus. All the representative images were taken from the middle turn of the cochlea. Scale bar is 20 μm.

Mentions: Multiple HDAC inhibitor studies have identified components regulated by HDACs of various anti-apoptotic pathways that underlie the neuroprotective effects found in models of inflammation, neurodegeneration, and oxidative stress.1–3,8–10 We used the information garnered from these HDAC inhibitor studies, and combined these data with what is known about the mechanism underlying ototoxic hair cell death to identify a potential mechanism of protection in the inner ear. HDAC inhibitors have primarily been thought to regulate histone acetylation and thus affect gene expression. However, HDACs have also been shown to regulate the acetylation of non-histone proteins including Forkhead Box O (FoxO) transcription factors, Sp1, and RelA/p65, and heat shock protein 90.4,5,22–24 Interestingly, a recent report found that during oxidative stress Foxo3a is regulated by HDAC2, which causes Foxo3a to switch from upregulating pro-survival factor Cdkn1a/p21CIP1 to upregulating the pro-apoptotic factor Bcl-2-like protein 11/Bim.23 During oxidative stress, HDAC inhibitor treatment blocks HDAC1-mediated deacetylation of Sp1 leading to hyperacetylation, which increases Sp1 DNA binding affinity causing an upregulation of pro-survival factor Cdkn1a/p21CIP1.3 Foxo3a and Sp1 have several common target genes that are co-regulated during cellular stress,25–27 and each of these transcription factors has been shown to switch between pro-apoptosis to pro-survival upon HDAC inhibitor treatment by upregulating pro-survival genes such as Cdkn1a/p21CIP1.3,23,27 To test whether Foxo3a- and Sp1-regulated genes are altered following SAHA and kanamycin treatment, we measured the expression levels of Cdkn1a/p21CIP1, Bcl2l11/Bim, and Hspa1a/Hsp70 by qPCR using Taqman probes on mRNA isolated from microdissected organ of Corti from wild-type mice using 100 mg/kg SAHA (FVB n = 8) and 600 mg/kg kanamycin (FVB n = 8) by our initial dosing schedule as described above (Figure 2h), but mice were euthanized 6 h after the last SAHA dose at P30. Our data show that the expression of both Cdkn1a/p21CIP1 and Hspa1a/Hsp70 are significantly increased in SAHA-treated mice compared with vehicle (DMSO) controls (Figure 5a). We also found a significant decrease in the expression of the pro-apoptosis factor Bcl2l11/Bim in SAHA-treated mice compared with vehicle (DMSO) controls (Figure 5a), suggesting a transcriptional switch in expression from pro-apoptosis to pro-survival genes.


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)

Nf-κB transcription factor RelA retains K310 acetylation resulting in increased expression of pro-survival genes. Taqman gene expression assays were done on microdissected organ of Corti from animals treated with a combination of SAHA/vehicle (DMSO) and kanamycin. Expression levels are relative to endogenous gene controls 18S, GAPDH, and Actb. Fold changes are shown relative to vehicle (DMSO)+kanamycin controls. (a) The expression levels of pro-survival genes Cflar, Bcl2l1, Cdkn1a, and Hspa1a were all increased significantly in SAHA+kanamycin-treated mice but the pro-apoptosis gene Bcl2l11 was significantly decreased compared with vehicle (DMSO) controls. Immunofluorescence of whole-mount organ of Corti was performed using antibodies against RelA/p65 K310 acetylation (red), Prestin (white), Pvalb (green), and Hoechst (blue). (b–e) We found that mice treated with SAHA and kanamycin retained RelA/65 K310 acetyl in the nucleus of the outer hair cells but mice treated with vehicle (DMSO) and kanamycin had a complete absence of RelA/65 K310 acetyl in the nucleus. All the representative images were taken from the middle turn of the cochlea. Scale bar is 20 μm.
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Related In: Results  -  Collection

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Figure 5: Nf-κB transcription factor RelA retains K310 acetylation resulting in increased expression of pro-survival genes. Taqman gene expression assays were done on microdissected organ of Corti from animals treated with a combination of SAHA/vehicle (DMSO) and kanamycin. Expression levels are relative to endogenous gene controls 18S, GAPDH, and Actb. Fold changes are shown relative to vehicle (DMSO)+kanamycin controls. (a) The expression levels of pro-survival genes Cflar, Bcl2l1, Cdkn1a, and Hspa1a were all increased significantly in SAHA+kanamycin-treated mice but the pro-apoptosis gene Bcl2l11 was significantly decreased compared with vehicle (DMSO) controls. Immunofluorescence of whole-mount organ of Corti was performed using antibodies against RelA/p65 K310 acetylation (red), Prestin (white), Pvalb (green), and Hoechst (blue). (b–e) We found that mice treated with SAHA and kanamycin retained RelA/65 K310 acetyl in the nucleus of the outer hair cells but mice treated with vehicle (DMSO) and kanamycin had a complete absence of RelA/65 K310 acetyl in the nucleus. All the representative images were taken from the middle turn of the cochlea. Scale bar is 20 μm.
Mentions: Multiple HDAC inhibitor studies have identified components regulated by HDACs of various anti-apoptotic pathways that underlie the neuroprotective effects found in models of inflammation, neurodegeneration, and oxidative stress.1–3,8–10 We used the information garnered from these HDAC inhibitor studies, and combined these data with what is known about the mechanism underlying ototoxic hair cell death to identify a potential mechanism of protection in the inner ear. HDAC inhibitors have primarily been thought to regulate histone acetylation and thus affect gene expression. However, HDACs have also been shown to regulate the acetylation of non-histone proteins including Forkhead Box O (FoxO) transcription factors, Sp1, and RelA/p65, and heat shock protein 90.4,5,22–24 Interestingly, a recent report found that during oxidative stress Foxo3a is regulated by HDAC2, which causes Foxo3a to switch from upregulating pro-survival factor Cdkn1a/p21CIP1 to upregulating the pro-apoptotic factor Bcl-2-like protein 11/Bim.23 During oxidative stress, HDAC inhibitor treatment blocks HDAC1-mediated deacetylation of Sp1 leading to hyperacetylation, which increases Sp1 DNA binding affinity causing an upregulation of pro-survival factor Cdkn1a/p21CIP1.3 Foxo3a and Sp1 have several common target genes that are co-regulated during cellular stress,25–27 and each of these transcription factors has been shown to switch between pro-apoptosis to pro-survival upon HDAC inhibitor treatment by upregulating pro-survival genes such as Cdkn1a/p21CIP1.3,23,27 To test whether Foxo3a- and Sp1-regulated genes are altered following SAHA and kanamycin treatment, we measured the expression levels of Cdkn1a/p21CIP1, Bcl2l11/Bim, and Hspa1a/Hsp70 by qPCR using Taqman probes on mRNA isolated from microdissected organ of Corti from wild-type mice using 100 mg/kg SAHA (FVB n = 8) and 600 mg/kg kanamycin (FVB n = 8) by our initial dosing schedule as described above (Figure 2h), but mice were euthanized 6 h after the last SAHA dose at P30. Our data show that the expression of both Cdkn1a/p21CIP1 and Hspa1a/Hsp70 are significantly increased in SAHA-treated mice compared with vehicle (DMSO) controls (Figure 5a). We also found a significant decrease in the expression of the pro-apoptosis factor Bcl2l11/Bim in SAHA-treated mice compared with vehicle (DMSO) controls (Figure 5a), suggesting a transcriptional switch in expression from pro-apoptosis to pro-survival genes.

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