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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.


Related in: MedlinePlus

Effects of HDAC inhibitors on apoptosis. (A) Cleaved caspase-3 staining in the neuromast from a control and TSA- or VPA-treated larva. White arrows indicate cells with cleaved caspase-3. Scale bar = 10 μm. (B) After treatment of larvae with 0.1 μM TSA or 100 μM VPA for 48 h, protein extracts were prepared and subjected to western blot assay using an antibody against cleaved caspase-3. β-Actin was included as the control.
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Figure 7: Effects of HDAC inhibitors on apoptosis. (A) Cleaved caspase-3 staining in the neuromast from a control and TSA- or VPA-treated larva. White arrows indicate cells with cleaved caspase-3. Scale bar = 10 μm. (B) After treatment of larvae with 0.1 μM TSA or 100 μM VPA for 48 h, protein extracts were prepared and subjected to western blot assay using an antibody against cleaved caspase-3. β-Actin was included as the control.

Mentions: Previous studies have suggested that HDAC inhibitors can induce cell cycle arrest and apoptosis in many different cell types (Riester et al., 2007). Because the reduced number of regenerated HCs might also be caused by increased cell death, we further monitored cell death using an antibody against cleaved caspase-3 over the time course of HC regeneration. We did not find any significant differences in cell death in the lateral line neuromasts between control and inhibitor-treated groups (Figure 7), and these results suggest that the inhibition of proliferation in HDAC inhibitor-treated neuromasts was mainly due to cell cycle arrest and not apoptosis.


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)

Effects of HDAC inhibitors on apoptosis. (A) Cleaved caspase-3 staining in the neuromast from a control and TSA- or VPA-treated larva. White arrows indicate cells with cleaved caspase-3. Scale bar = 10 μm. (B) After treatment of larvae with 0.1 μM TSA or 100 μM VPA for 48 h, protein extracts were prepared and subjected to western blot assay using an antibody against cleaved caspase-3. β-Actin was included as the control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Effects of HDAC inhibitors on apoptosis. (A) Cleaved caspase-3 staining in the neuromast from a control and TSA- or VPA-treated larva. White arrows indicate cells with cleaved caspase-3. Scale bar = 10 μm. (B) After treatment of larvae with 0.1 μM TSA or 100 μM VPA for 48 h, protein extracts were prepared and subjected to western blot assay using an antibody against cleaved caspase-3. β-Actin was included as the control.
Mentions: Previous studies have suggested that HDAC inhibitors can induce cell cycle arrest and apoptosis in many different cell types (Riester et al., 2007). Because the reduced number of regenerated HCs might also be caused by increased cell death, we further monitored cell death using an antibody against cleaved caspase-3 over the time course of HC regeneration. We did not find any significant differences in cell death in the lateral line neuromasts between control and inhibitor-treated groups (Figure 7), and these results suggest that the inhibition of proliferation in HDAC inhibitor-treated neuromasts was mainly due to cell cycle arrest and not apoptosis.

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