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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 the expression of p21Cip1, p27Kip1, and p53 protein. 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 antibodies against p21Cip1, p27Kip1, and p53. β-Actin was included as the control. Mean ± s.e.m. for three experimental replicates. *p < 0.05.
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Figure 6: Effects of HDAC inhibitors on the expression of p21Cip1, p27Kip1, and p53 protein. 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 antibodies against p21Cip1, p27Kip1, and p53. β-Actin was included as the control. Mean ± s.e.m. for three experimental replicates. *p < 0.05.

Mentions: HDAC plays a central role in the regulation of cell cycles (Xiao et al., 2014). We investigated the effect of HDACs on the expression of cell cycle-regulated proteins during the recovery period. The results revealed that HDAC inhibition significantly decreased the expressions of cyclin B1, cyclin D1, cyclin D3, cyclin E1, cyclin-dependent kinase (CDK) 2, and CDC2, whereas increased the expression of p21 and p27 (Supplemental Figure 2). These results suggest that HDAC inhibitors might inhibit the proliferation of neuromast cells by inducing p21 and p27, leading to G1 phase cell cycle arrest. To further confirm the effect of HDAC inhibition on p21Cip1 and p27Kip1 expression during the recovery period, embryos were treated with TSA or VPA after neomycin-induced injury and protein expression was detected using antibodies specific to p21Cip1 and p27Kip1. The levels of p21Cip1 and p27Kip1 in the inhibitor-treated groups increased compared to controls (Figure 6). Because it is well-known that the p21Cip1 gene is physiologically induced by p53 (Sherr, 1994), we next examined the expression of p53 by western blotting. The expression of p53 was not significantly altered in fish treated with TSA or VPA compared to controls, and this suggests that the induction of p21Cip1 expression in response to HDAC inhibitors might be mediated by a p53-independent pathway.


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 the expression of p21Cip1, p27Kip1, and p53 protein. 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 antibodies against p21Cip1, p27Kip1, and p53. β-Actin was included as the control. Mean ± s.e.m. for three experimental replicates. *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Effects of HDAC inhibitors on the expression of p21Cip1, p27Kip1, and p53 protein. 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 antibodies against p21Cip1, p27Kip1, and p53. β-Actin was included as the control. Mean ± s.e.m. for three experimental replicates. *p < 0.05.
Mentions: HDAC plays a central role in the regulation of cell cycles (Xiao et al., 2014). We investigated the effect of HDACs on the expression of cell cycle-regulated proteins during the recovery period. The results revealed that HDAC inhibition significantly decreased the expressions of cyclin B1, cyclin D1, cyclin D3, cyclin E1, cyclin-dependent kinase (CDK) 2, and CDC2, whereas increased the expression of p21 and p27 (Supplemental Figure 2). These results suggest that HDAC inhibitors might inhibit the proliferation of neuromast cells by inducing p21 and p27, leading to G1 phase cell cycle arrest. To further confirm the effect of HDAC inhibition on p21Cip1 and p27Kip1 expression during the recovery period, embryos were treated with TSA or VPA after neomycin-induced injury and protein expression was detected using antibodies specific to p21Cip1 and p27Kip1. The levels of p21Cip1 and p27Kip1 in the inhibitor-treated groups increased compared to controls (Figure 6). Because it is well-known that the p21Cip1 gene is physiologically induced by p53 (Sherr, 1994), we next examined the expression of p53 by western blotting. The expression of p53 was not significantly altered in fish treated with TSA or VPA compared to controls, and this suggests that the induction of p21Cip1 expression in response to HDAC inhibitors might be mediated by a p53-independent pathway.

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