UCHL1 regulated by Sp1 ameliorates cochlear hair cell senescence and oxidative damage

Abstract

Age-related hearing loss (ARHL) is the most common cause of hearing loss in the elderly. Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme involved in several types of human disease. The present study aimed to investigate the effect of UCHL1 on a hydrogen peroxide (H₂O₂)-induced ARHL model in cochlear hair cells and uncover its underlying mechanism. Reverse transcription-quantitative (RT-q)PCR and western blot analysis were used to assess UCHL1 expression in HEI-OC1 cells exposed to H₂O₂. Following UCHL1 overexpression in H₂O₂-induced HEI-OC1 cells, cell activity was assessed by Cell Counting Kit-8 assay. The content of oxidative stress-associated markers including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and reactive oxygen species (ROS ) was measured using corresponding commercial kits. Cell apoptosis was evaluated by TUNEL assay and western blot analysis. Cell senescence was assessed by senescence-associated β-galactosidase staining and western blot analysis. RT-qPCR and western blot analysis were applied to measure mRNA and protein expression levels, respectively, of specificity protein 1 (Sp1) in H₂O₂-treated HEI-OC1 cells. In addition, the association between UCHL1 and Sp1 was verified by luciferase reporter and chromatin immunoprecipitation (ChIP) assay. The mRNA and protein expression levels of UCHL1 were also determined in Sp1-overexpressing cells by RT-qPCR and western blot analysis, respectively. Following Sp1 overexpression in UCHL1-overexpressing H₂O₂-treated HEI-OC1 cells, cell activity, oxidative stress, apoptosis and senescence were assessed. Finally, the expression levels of NF-κB signaling-related proteins p-NF-κB p65 and NF-κB p65 were detected using western blot analysis. The results showed that UCHL1 was downregulated in H₂O₂-treated HEI-OC1 cells. In addition, UCHL1 overexpression enhanced cell viability and promoted oxidative damage, apoptosis and senescence in H₂O₂-induced HEI-OC1 cells. Furthermore, Sp1 was upregulated in H₂O₂-treated HEI-OC1 cells. Additionally, luciferase reporter and ChIP assays demonstrated that Sp1 interacted with the UCHL1 promoter to inhibit UCHL1 transcription. Sp1 overexpression reversed the effect of UCHL1 overexpression on cell viability, oxidative stress, apoptosis, senescence and activation of the NF-κB signaling pathway in H₂O_2-exposed HEI-OC1 cells. Collectively, the results suggested that UCHL1 transcriptional suppression by Sp1 protected cochlear hair cells from H₂O₂-triggered senescence and oxidative damage.

Keywords: age-related hearing loss; oxidative stress; senescence; specificity protein 1; ubiquitin carboxyl-terminal hydrolase L1.

Figure 1

UCHL1 elevation improves the viability of H₂O₂-treated HEI-OC1 cells. (A) RT-qPCR and (B) western blot analysis of UCHL1 expression in H₂O₂-insulted HEI-OC1 cells. Analysis of overexpression efficacy of pcDNA3.1-UCHL1 recombinant plasmid via (C) RT-qPCR and (D) western blotting. (E) Viability of H₂O₂-exposed HEI-OC1 cells was evaluated by Cell Counting Kit-8 assay. ^**P<0.01 and ^***P<0.001 vs. control. ^#P<0.05 vs. H₂O₂ + Oe-NC. UCHL1, ubiquitin carboxyl-terminal hydrolase L1; RT-qPCR, reverse transcription-quantitative PCR; H₂O₂, hydrogen peroxide; Oe-NC, overexpression negative control.

Figure 2

UCHL1 upregulation mitigates H₂O₂-mediated oxidative injury and apoptosis in HEI-OC1 cells. (A) Detection of levels of oxidative stress markers using the corresponding kits. (B) TUNEL assay estimated the apoptosis of H₂O₂-exposed HEI-OC1 cells. Magnification, x200. (C) Quantification of cell apoptotic rate. (D) Western blot analysis of expression of apoptosis-associated factors. ^***P<0.001 vs. control. ^##P<0.01 and ^###P<0.001 vs. H₂O₂ + Oe-NC. SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; ROS, reactive oxygen species; Bcl-2, B cell lymphoma-2; H₂O₂, hydrogen peroxide; Oe-NC, overexpression negative control.

Figure 3

Overexpression of UCHL1 halts H₂O₂-triggered HEI-OC1 cell senescence. (A) SA-β-gal staining indicating cell senescence. Magnification, x100. (B) Western blot analysis of expression of senescence-associated factors. ^***P<0.001 vs. control. ^###P<0.001 vs. H₂O₂ + Oe-NC. UCHL1, ubiquitin carboxyl-terminal hydrolase L1; H₂O₂, hydrogen peroxide; Oe-NC, overexpression negative control; SA-β-gal, senescence-associated β-galactosidase.

Figure 4

Sp1 suppresses transcription of UCHL1. (A) RT-qPCR and (B) western blot analysis Sp1 expression in H₂O₂-insulted HEI-OC1 cells. Analysis of overexpression efficacy of Oe-Sp1 plasmid by (C) RT-qPCR and (D) western blotting. ^***P<0.001 vs. control. (E) Luciferase reporter assay verified the luciferase activity of UCHL1-WT and UCHL1-MUT. ^***P<0.001 vs. Oe-NC. (F) Chromatin immunoprecipitation assay identified the accumulation of UCHL1 promoter in Sp1 antibody. ^***P<0.001 vs. input. (G) RT-qPCR and (H) western blot analysis of UCHL1 expression after Sp1 was overexpressed. ^***P<0.001 vs. control. UCHL1, ubiquitin carboxyl-terminal hydrolase L1; Sp1, specificity protein 1; RT-q, reverse transcription-quantitative; H₂O₂, hydrogen peroxide; Oe-NC, overexpression negative control; WT, wild-type; MUT, mutant.

Figure 5

Overexpression of Sp1 abrogates the protective role of UCHL1 in H₂O₂-induced HEI-OC1 cell injury. (A) Viability of H₂O₂-exposed HEI-OC1 cells evaluated by CCK-8 assay. (B) Detection of the levels of oxidative stress markers using corresponding kits. (C) TUNEL assay of apoptosis of H₂O₂-exposed HEI-OC1 cells. Magnification, x200. (D) Western blot analysis of expression of apoptosis-associated factors. (E) SA-β-gal staining analysis of cell senescence. Western blot analysis of expression of (F) senescence- and (G) NF-κB signaling-associated factors. ^***P<0.001 vs. control. ^##P<0.01 and ^###P<0.001 vs. H₂O₂. ^∆P<0.05, ^∆∆P<0.01 and ^∆∆∆P<0.001 vs. H₂O₂ + Oe-UCHL1 + Oe-NC. UCHL1, ubiquitin carboxyl-terminal hydrolase L1; Sp1, specificity protein 1; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; ROS, reactive oxygen species; Bcl-2, B cell lymphoma-2; p-, phosphorylated; Oe-NC, negative control; SA-β-gal, senescence-associated β-galactosidase.