Short exposure to sodium arsenite (NaAsO2) induced a premature senescence phenotype in HEI-OC1 cells
Senescent cells are in a state of permanent cell cycle arrest and remain viable, unlike apoptotic cells. First, based on prior reports on cellular senescence [19, 24] and concentrations of NaAsO2 [22, 43], we assessed the cell proliferation rate and viability after auditory cells were exposed to medium containing different concentrations of NaAsO2 (125, 250 and 500 µM) for 1 h and then incubated in complete medium without NaAsO2 for 3 days. The cell proliferation rate of cells subjected to a short exposure to NaAsO2 decreased in a dose- and time-dependent manner (Fig. 1A), while the cell viability was unchanged, as the ratio of dead cells remained below 3% even 3 days after a short exposure to NaAsO2 (Fig. 1B). We regarded this condition as a model of NaAsO2-induced premature senescence in auditory cells.
Next, we investigated the phosphorylation of histone H2AX (γH2AX) as a trigger of the DNA damage response (DDR) in auditory cells to confirm whether NaAsO2 induces double-strand breaks (DSBs) [44]. γH2AX is an indicator of DSBs and an initiator of the DDR [45]. Immunofluorescence staining analysis indicated that the number of γH2AX foci/cell was significantly higher 24 h after exposure to NaAsO2 than that after exposure to vehicle in HEI-OC1 cells (Fig. S2A). This result suggested that the DDR caused by a short NaAsO2 exposure might trigger the induction of premature senescence in auditory cells.
We observed the expression of the cyclin-dependent kinases p21 (cdkn1a) and p16 (cdkn2a) to investigate whether NaAsO2 exposure induces cell cycle arrest in auditory cells (Fig. S2B; right panel: mRNA level, left panel: protein level; S1A, full-length blots). The expression of p21 was increased and peaked at 12 h, while p16 expression did not change. This result indicated that permanent cell cycle arrest in G1 or G2-M phase occurred in cells subjected to a short exposure to NaAsO2 [46]. Finally, we evaluated the positive rate of senescence-associated βgalactosidase (SA-βgal), which is widely used as a cellular senescence marker, after a short exposure to NaAsO2 and the expression of senescence-associated secretory phenotype (SASP) genes (IL-6, IL-1β and cxcl10), which induce senescence in surrounding cells or re-enforce senescent cells themselves as paracrine factors to confirm the induction of the senescence phenotype in auditory cells. SA-βgal-positive cell numbers were significantly increased at 3 days after a short NaAsO2 exposure (Fig. 1C), and the cells showed an enlarged, flattened and irregular morphology, which are regarded as characteristics of senescent cells (Fig. S2C, left panel). Positive staining for SA-βgal was mainly observed in these cells. The mean areas of SA-βgal-positive cells were significantly wider than those of SA-βgal-negative cells (Fig. S1C, right panel).
The expression of SASP-related genes (IL-6, IL-1β and cxcl10) was also significantly elevated in cells subjected to a short NaAsO2 (Fig. 1D). These findings indicated that the senescence phenotype was initiated by the DDR due to short exposure to NaAsO2 in auditory cells.
Intracellular ROS derived from damaged mitochondria contribute to NaAsO 2 -induced premature senescence as a second messenger in auditory cells
We evaluated whether exposure to NaAsO2 increases intracellular ROS levels derived from damaged mitochondria in HEI-OC1 cells because NaAsO2 functions as a second messenger that induces premature senescence in auditory cells, indicating mitochondrial dysfunction [27, 47]. As shown in Fig. 2A upper panel, ROS levels were extremely elevated in a dose-dependent manner at 6 h after a short exposure to NaAsO2 and higher than those observed with 800 µM H2O2, which was used as a positive control; however, this effect was completely suppressed by N-acetylcysteine (NAC), a well-known radical scavenger that has been used to modulate oxidative stress (Fig. 2A lower panel). Importantly, NAC treatment released auditory cells from cell cycle arrest by suppressing the expression of p21 (Figs. 2B; S1B, full-length blots) and led to a decrease in SA-βgal-positive cell numbers in cells with NaAsO2-induced premature senescence (Fig. 2C). These results indicate that intracellular ROS derived from damaged mitochondria regulate the cell cycle in auditory cells under oxidative stress, inducing premature senescence due to mitochondrial dysfunction separate from the DDR.
Short NaAsO 2 exposure leads to reduced degradation capacity of the autophagy‒lysosome pathway (APL), affecting the induction of premature senescence in HEI-OC1 cells
Based on our previous report [19], we considered that oxidative stress-induced autophagy serves as a protective mechanism against premature senescence in auditory cells; we investigated the induction of LC3, which is a marker of autophagy, and p62, which turns over through autophagic degradation, and measured autophagic flux, an index of the autophagy degradation ability, in HEI-OC1 cells subjected to a short exposure to NaAsO2 to confirm whether autophagy is activated as a part of the DDR and regulates NaAsO2-induced cellular senescence in auditory cells. The expression of LC3-Ⅱ and p62 was increased in a dose-dependent manner (Fig. 3A left panel) (Fig. S1C) and peaked at 24 h (Fig. 3A right panel) (Fig. S1C). As shown in Fig. S1D and S3A), the expression of LC3-Ⅱ and p62 in bafilomycin A1 (BafA1)-treated cells was increased more at 24 h after a short exposure to NaAsO2 than after nonexposed cells. This result indicates that autophagic flux was promoted at 24 h after a short NaAsO2 exposure, which means that autophagic degradation activity occurs at this point in auditory cells.
Next, we considered the expression of lysosome-associated membrane protein 1 (LAMP1) and Lysotracker®Red DND-99, which are used as indicators of lysosomal abundance, via immunostaining in cells subjected to a short exposure to NaAsO2. LAMP1 is expressed on the surface of different organelles, including lysosomes, autolysosomes, endosomes, multivesicular bodies and multilamellar bodies (MVBs), in the cytoplasm [48, 49]. The fluorescence intensity of LAMP1 (Fig. 3B, left panel) was obviously elevated at 24 h after a short NaAsO2 exposure. It was higher than that of the EBSS-starved control. This result suggested that there might be LAMP1 positive-degradative and nondegradative organelles at this point in cells subjected to a short exposure to NaAsO2, which means there is an abundance of lysosomes.
According to previous reports, LysoTracker selectively accumulates in acidic organelles, including acidic vesicles, late endosomes and lysosomes [50, 51]. Lysotracker®Red DND-99 (Fig. 3B, right panel) staining was also significantly increased 24 h after a short NaAsO2 exposure. This immunoexpression level was higher than that of EBSS, which was used as a positive control. This result indicated that lysosomal pH maintenance was compromised by a short NaAsO2 exposure. Namely, impaired organelles due to damaged lysosomes might accumulate in the cytoplasm of cells exhibiting auditory senescence at this point, with the lysosomal pH being maintained in an acidic environment.
Next, we investigated the time course of the TFEB-mediated gene expression of autophagy-related genes (LC3B and p62) and lysosome-related genes, including lysosome-associated membrane protein 1 (LAMP1) and representative proteases in lysosomes (Cathepsin B and Cathepsin D), in cells subjected to a short exposure to NaAsO2 by performing RT‒qPCR to evaluate the transcriptional function of TFEB and confirm the time-dependent changes in the autophagy-lysosomal pathway after a short exposure to NaAsO2.
As shown in Fig. 3C (left panel), the expression of autophagy-related genes was increased with a peak of 6 h, and that of lysosome-related genes was increased with a peak of 3 h. The expression level at 6 h was almost the same as that under EBSS starvation (Fig. 3C, right panel). Namely, the autophagy‒lysosome pathway (ALP) was activated at the early stage to protect auditory cells from NaAsO2-induced premature senescence, but it was only temporary, and the degradation capacity decreased soon after. These results indicate that the NaAsO2-induced impairment of autophagic degradation in senescent auditory cells is closely related to the reduced degradation capacity of the autophagy‒lysosome pathway (APL).
Next, we investigated the effect of TFEB on lysosomal function during premature senescence in auditory cells after a short exposure to NaAsO2. Lysosomal impairment induced by treatment with BafA1, which inhibits vacuolar ATPase (v-ATPase) on the lysosomal membrane, promotes alkalinization of the lysosomal lumen, and chloroquine (CQ), which elevates the lysosomal pH and inhibits lysosomal degradation, significantly increased the ratio of SA-βgal-positive cells among auditory cells after a short exposure to NaAsO2 by blocking the fusion of autophagosomes with lysosomes and impairing autophagic degradation through the autophagy‒lysosome pathway (APL) (Fig. S3B) [52, 53]. These results indicate that TFEB-associated lysosomal dysfunction directly affects the induction of premature senescence by blocking the fusion of autophagosomes with lysosomes in NaAsO2-exposed auditory cells.
We investigated the impact of TFEB on autophagy in auditory cells with premature senescence after a short exposure to NaAsO2. The mTOR inhibitor rapamycin obviously decreased the ratio of SA-βgal-positive cells among auditory cells after a short exposure to NaAsO2 by promoting autophagic degradation (Fig. S3C). This result means that the regulation of TFEB-mediated autophagy could directly affect premature senescence. Next, we examined ultrastructural premature senescence in NaAsO2-exposed auditory cells (Fig. S3D1-8). Importantly, ultrastructure analysis under TEM indicated that damaged mitochondria (Fig. S3D3-4) or dense organelles within immature autophagosomes, autolysosomes and multivesicular bodies (MVBs) or aggregates (Fig. S3D5-6) accumulated at 24 h in the cytoplasm of NaAsO2-exposed auditory cells [54]. Other autophagosomes eventually appeared to merge with lysosomes to become autolysosomes containing partially degraded material that appeared as electron-dense, unevenly distributed dense masses (Fig. S3D5-6). On the other hand, autophagosomes in EBSS-starved cells had a perfect shape with round, double-membraned structures (Fig. S3D7-8). This result means that the autophagy‒lysosome pathway (ALP) was defective at the ultrastructural level in NaAsO2-exposed auditory cells.
TFEB nuclear translocation in auditory cells after exposure to NaAsO2 was only temporary
We focused on the function of transcription factor EB (TFEB), evaluating a TFEB nuclear export signal that activates autophagy and regulates lysosomal biogenesis in HEI-OC1 cells after a short exposure to NaAsO2 [28, 30, 55]. The expression of TFEB was increased at the peak of 3 h after a short exposure to NaAsO2 in the nuclear fraction but increased after 6 h in the cytoplasmic fraction (Figs. 4A and S1E, full-length blots), and the localization of TFEB in the nucleus was also significantly increased at 3 h (Fig. 4B). TFEB nuclear translocation was induced in a dose-dependent manner in cells subjected to a short exposure to NaAsO2 (Figs. S4 and S1F, full-length blots). These results indicate that endogenous TFEB in auditory cells is translocated into the nucleus from the cytoplasm and became temporarily activated until 3 h in a dose-dependent manner after a short exposure to NaAsO2 but subsequently declines.
TFEB directly controls the induction of premature senescence by regulating the autophagy lysosomal pathway (ALP) and ROS derived from damaged mitochondria in HEI-OC1 cells subjected to a short exposure to NaAsO 2
Finally, we knocked down TFEB expression using two different short interfering RNAs (siRNAs; #1: Santa Cruz Biotechnology, Inc., CA, USA and #2: Dharmacon Technologies, Lafayette, CO, USA) (Fig. S5) and evaluated the transcriptional function of TFEB during the induction of the premature senescence phenotype in HEI-OC1 cells after a short exposure to NaAsO2. Interestingly, the ratio of SA-βgal-positive cells (Fig. 5A) and the expression of SASP-related genes (IL-6, IL1, and CXCL10) (Fig. 5B) significantly increased in TFEB KD HEI-OC1 cells after a short exposure to NaAsO2. This result suggests that TFEB directly controls the induction of the premature senescence phenotype in cells subjected to a short exposure to NaAsO2.
Next, we investigated the transcriptional effects of TFEB-mediated targeting of autophagy-lysosomal pathway genes on premature senescence in auditory cells after a short exposure to NaAsO2. The expression of autophagy-related genes (LC3B and p62) (Fig. 5C) and lysosome-related genes (LAMP1, cathepsin B and cathepsin D) (Fig. 5D) was significantly decreased in TFRB KD cells. This result indicates that TFEB promotes autophagy and lysosomal biogenesis in auditory cells, regulating the transcription of autophagy-related genes (LC3B and p62) and lysosome-related genes (LAMP1, cathepsin B and cathepsin D) at the transcriptional level.
As shown in Fig. 5A-D, the induction of a premature senescence phenotype and the expression of TFEB target genes were negatively correlated in auditory cells after a short exposure to NaAsO2. These results suggested that TFEB targeting ALP dysfunction leads to premature senescence in auditory cells subjected to a short exposure to NaAsO2.
As shown in Figs. S6 and S1G (full-length blots), NAC substantially decreased the expression of TFEB in the nuclear fraction but increased TFEB expression in the cytoplasmic fraction. Interestingly, the knockdown of TFEB via siRNA significantly increased the production of ROS from damaged mitochondria in NaAsO2-exposed cells (Fig. 5E). These two results mean that the TFEB export signal is dependent on ROS production derived from damaged mitochondria, controlling mitochondrial quality.