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Effect of histone deacetylase inhibitors trichostatin A and valproic acid on hair cell regeneration in zebrafish lateral line neuromasts.

He Y, Cai C, Tang D, Sun S, Li H - Front Cell Neurosci (2014)

Bottom Line: Additionally, BrdU immunostaining and western blot analysis showed that TSA or VPA treatment caused a significant decrease in the percentage of S-phase cells and induced p21(Cip1) and p27(Kip1) expression, both of which are likely to explain the decrease in the amount of newly regenerated hair cells in treated embryos.Finally, we showed that HDAC inhibitors induced no observable cell death in neuromasts as measured by cleaved caspase-3 immunohistochemistry and western blot analysis.Taken together, our results demonstrate that HDAC activity has an important role in the regeneration of hair cells in the lateral line.

View Article: PubMed Central - PubMed

Affiliation: Department of Otorhinolaryngology, Affiliated Eye and ENT Hospital of Fudan University Shanghai, China.

ABSTRACT
In humans, auditory hair cells are not replaced when injured. Thus, cochlear hair cell loss causes progressive and permanent hearing loss. Conversely, non-mammalian vertebrates are capable of regenerating lost sensory hair cells. The zebrafish lateral line has numerous qualities that make it well-suited for studying hair cell development and regeneration. Histone deacetylase (HDAC) activity has been shown to have an important role in regenerative processes in vertebrates, but its function in hair cell regeneration in vivo is not fully understood. Here, we have examined the role of HDAC activity in hair cell regeneration in the zebrafish lateral line. We eliminated lateral line hair cells of 5-day post-fertilization larvae using neomycin and then treated the larvae with HDAC inhibitors. To assess hair cell regeneration, we used 5-bromo-2-deoxyuridine (BrdU) incorporation in zebrafish larvae to label mitotic cells after hair cell loss. We found that pharmacological inhibition of HDACs using trichostatin A (TSA) or valproic acid (VPA) increased histone acetylation in the regenerated neuromasts following neomycin-induced damage. We also showed that treatment with TSA or VPA decreased the number of supporting cells and regenerated hair cells in response to hair cell damage. Additionally, BrdU immunostaining and western blot analysis showed that TSA or VPA treatment caused a significant decrease in the percentage of S-phase cells and induced p21(Cip1) and p27(Kip1) expression, both of which are likely to explain the decrease in the amount of newly regenerated hair cells in treated embryos. Finally, we showed that HDAC inhibitors induced no observable cell death in neuromasts as measured by cleaved caspase-3 immunohistochemistry and western blot analysis. Taken together, our results demonstrate that HDAC activity has an important role in the regeneration of hair cells in the lateral line.

No MeSH data available.


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HDAC inhibition increases histone acetylation during HC regeneration. (A) Western blot analysis of acetylated H3 (Ace H3) and acetylated H4 (Ace H4) protein extracts from control and 0.1 μM TSA- or 100 μM VPA-treated larvae at 48 h after neomycin damage. β-Actin was included as the control. Mean ± s.e.m. for three experimental replicates. *p < 0.05. **p < 0.001. (B) Acetylation of histone H3 and H4 were stained (red) by Ace H3 and Ace H4 antibody. HCs were labeled with HCS-1 (green) antibody and nuclei were stained with DAPI (blue). Scale bar = 10 μm.
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Figure 8: HDAC inhibition increases histone acetylation during HC regeneration. (A) Western blot analysis of acetylated H3 (Ace H3) and acetylated H4 (Ace H4) protein extracts from control and 0.1 μM TSA- or 100 μM VPA-treated larvae at 48 h after neomycin damage. β-Actin was included as the control. Mean ± s.e.m. for three experimental replicates. *p < 0.05. **p < 0.001. (B) Acetylation of histone H3 and H4 were stained (red) by Ace H3 and Ace H4 antibody. HCs were labeled with HCS-1 (green) antibody and nuclei were stained with DAPI (blue). Scale bar = 10 μm.

Mentions: To confirm that the deficiency in HC regeneration was, indeed, caused by the inhibition of HDAC activity, we examined the level of histone acetylation in zebrafish after treatment with 0.1 μM TSA or 100 μM VPA for 48 h. Western blot analysis showed that the levels of acetylated histone H3 and H4 in controls were low, but incubation with TSA and VPA resulted in the accumulation of acetylated histones H3 and H4 (Figure 8A). To more accurately localize acetylated H3 and H4 expression to the specific cell types in the neuromast, we performed immunostaining for the HC marker HCS-1 (green). In controls, both acetylated H3+ (red) and acetylated H4+ (red) cells are expressed outside the central HCs and there was little or no overlap of signals (Figure 8B). However, the acetylated H3 and H4 signals were elevated in inhibitor-treated neuromast cells, particularly in the SCs, and more overlap was seen between the signals (Figure 8B). These results provide evidence that HDAC inhibitor-mediated histone acetylation in neuromast cells might indeed be responsible for the decreased HC production. We next investigated which members of HDAC family are affected by HDAC inhibitors treatment during the recovery period. We found a significant downregulation of HDAC1, HDAC3, and HDAC4 protein levels in zebrafish treated with the VPA or TSA. Reduction of HDAC2 protein levels is also found after 48 h HDAC inhibitor treatment. Furthermore, VPA and TSA treatment do not cause a reduction in protein levels of HDACs 5, 6, and 8 (Supplemental Figure 3).


Effect of histone deacetylase inhibitors trichostatin A and valproic acid on hair cell regeneration in zebrafish lateral line neuromasts.

He Y, Cai C, Tang D, Sun S, Li H - Front Cell Neurosci (2014)

HDAC inhibition increases histone acetylation during HC regeneration. (A) Western blot analysis of acetylated H3 (Ace H3) and acetylated H4 (Ace H4) protein extracts from control and 0.1 μM TSA- or 100 μM VPA-treated larvae at 48 h after neomycin damage. β-Actin was included as the control. Mean ± s.e.m. for three experimental replicates. *p < 0.05. **p < 0.001. (B) Acetylation of histone H3 and H4 were stained (red) by Ace H3 and Ace H4 antibody. HCs were labeled with HCS-1 (green) antibody and nuclei were stained with DAPI (blue). Scale bar = 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4230041&req=5

Figure 8: HDAC inhibition increases histone acetylation during HC regeneration. (A) Western blot analysis of acetylated H3 (Ace H3) and acetylated H4 (Ace H4) protein extracts from control and 0.1 μM TSA- or 100 μM VPA-treated larvae at 48 h after neomycin damage. β-Actin was included as the control. Mean ± s.e.m. for three experimental replicates. *p < 0.05. **p < 0.001. (B) Acetylation of histone H3 and H4 were stained (red) by Ace H3 and Ace H4 antibody. HCs were labeled with HCS-1 (green) antibody and nuclei were stained with DAPI (blue). Scale bar = 10 μm.
Mentions: To confirm that the deficiency in HC regeneration was, indeed, caused by the inhibition of HDAC activity, we examined the level of histone acetylation in zebrafish after treatment with 0.1 μM TSA or 100 μM VPA for 48 h. Western blot analysis showed that the levels of acetylated histone H3 and H4 in controls were low, but incubation with TSA and VPA resulted in the accumulation of acetylated histones H3 and H4 (Figure 8A). To more accurately localize acetylated H3 and H4 expression to the specific cell types in the neuromast, we performed immunostaining for the HC marker HCS-1 (green). In controls, both acetylated H3+ (red) and acetylated H4+ (red) cells are expressed outside the central HCs and there was little or no overlap of signals (Figure 8B). However, the acetylated H3 and H4 signals were elevated in inhibitor-treated neuromast cells, particularly in the SCs, and more overlap was seen between the signals (Figure 8B). These results provide evidence that HDAC inhibitor-mediated histone acetylation in neuromast cells might indeed be responsible for the decreased HC production. We next investigated which members of HDAC family are affected by HDAC inhibitors treatment during the recovery period. We found a significant downregulation of HDAC1, HDAC3, and HDAC4 protein levels in zebrafish treated with the VPA or TSA. Reduction of HDAC2 protein levels is also found after 48 h HDAC inhibitor treatment. Furthermore, VPA and TSA treatment do not cause a reduction in protein levels of HDACs 5, 6, and 8 (Supplemental Figure 3).

Bottom Line: Additionally, BrdU immunostaining and western blot analysis showed that TSA or VPA treatment caused a significant decrease in the percentage of S-phase cells and induced p21(Cip1) and p27(Kip1) expression, both of which are likely to explain the decrease in the amount of newly regenerated hair cells in treated embryos.Finally, we showed that HDAC inhibitors induced no observable cell death in neuromasts as measured by cleaved caspase-3 immunohistochemistry and western blot analysis.Taken together, our results demonstrate that HDAC activity has an important role in the regeneration of hair cells in the lateral line.

View Article: PubMed Central - PubMed

Affiliation: Department of Otorhinolaryngology, Affiliated Eye and ENT Hospital of Fudan University Shanghai, China.

ABSTRACT
In humans, auditory hair cells are not replaced when injured. Thus, cochlear hair cell loss causes progressive and permanent hearing loss. Conversely, non-mammalian vertebrates are capable of regenerating lost sensory hair cells. The zebrafish lateral line has numerous qualities that make it well-suited for studying hair cell development and regeneration. Histone deacetylase (HDAC) activity has been shown to have an important role in regenerative processes in vertebrates, but its function in hair cell regeneration in vivo is not fully understood. Here, we have examined the role of HDAC activity in hair cell regeneration in the zebrafish lateral line. We eliminated lateral line hair cells of 5-day post-fertilization larvae using neomycin and then treated the larvae with HDAC inhibitors. To assess hair cell regeneration, we used 5-bromo-2-deoxyuridine (BrdU) incorporation in zebrafish larvae to label mitotic cells after hair cell loss. We found that pharmacological inhibition of HDACs using trichostatin A (TSA) or valproic acid (VPA) increased histone acetylation in the regenerated neuromasts following neomycin-induced damage. We also showed that treatment with TSA or VPA decreased the number of supporting cells and regenerated hair cells in response to hair cell damage. Additionally, BrdU immunostaining and western blot analysis showed that TSA or VPA treatment caused a significant decrease in the percentage of S-phase cells and induced p21(Cip1) and p27(Kip1) expression, both of which are likely to explain the decrease in the amount of newly regenerated hair cells in treated embryos. Finally, we showed that HDAC inhibitors induced no observable cell death in neuromasts as measured by cleaved caspase-3 immunohistochemistry and western blot analysis. Taken together, our results demonstrate that HDAC activity has an important role in the regeneration of hair cells in the lateral line.

No MeSH data available.


Related in: MedlinePlus