FAM134B-induced endoplasmic reticulum (ER)-phagy exacerbates cisplatin-insulted hair cell apoptosis ：Possible relation to excessive ER stress.

AIMS: FAM134B, the initial endoplasmic reticulum (ER)-phagy receptor identified, facilitates ER-phagy during ER stress. The malfunction of FAM134B has been demonstrated to have a crucial role in the pathological mechanisms of diverse human ailments. However, the role of FAM134B-mediated ER-phagy in ototoxicity, particularly in cisplatin-induced ototoxicity, remains unclear. The present study endeavors to investigate whether FAM134B is expressed in House Ear Institute-Organ of Corti 1 (HEI-OC1) and C57BL/6 murine cochlear hair cells (HCs), and to explore its potential function in cisplatin-mediated ototoxicity, with the aim of discovering new insights that can mitigate or forestall the irreversible adverse effect of cisplatin. METHODS: Immunofluorescence (IF) staining was used to test the expression pattern of FAM134B, levels of C/EBP-homologous protein (CHOP), autophagy, and co-localization ratio of lysosomes and ER. Western blotting was employed to measure changes in expression levels of FAM134B, LC3B, ER stress-related proteins, LAMP1 and apoptotic mediators. Cell apoptosis was examined using transferase dUTP nick end labeling (TUNEL) assay and flow cytometry. RESULTS: In the present investigation, it was observed that FAM134B exhibited a diffuse expression pattern in the cytoplasm and nuclei of control HEI-OC1 cells. Following cisplatin administration, FAM134B was found to accumulate and form distinct dots around the nuclei, concomitant with increased levels of ER-phagy, ER stress, unfolded protein response (UPR), and cell apoptosis. Additionally, knockdown of FAM134B resulted in reduced ER-phagy, mitigated ER stress and UPR, and decreased apoptotic activity in HEI-OC1 cells following cisplatin exposure. CONCLUSIONS: Collectively, the findings of this study demonstrate that FAM134B-mediated ER-phagy enhances the susceptibility of HCs to ER stress and apoptosis in response to cisplatin-induced stress. This suggests a sequential progression of ER-phagy, ER stress and apoptosis following cisplatin stimulus, and implies the potential therapeutic benefit of inhibiting of FAM134B-mediated ER-phagy in the prevention of cisplatin-related ototoxicity.

2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) activates Aryl hydrocarbon receptor (AhR) mediated ROS and NLRP3 inflammasome/p38 MAPK pathway inducing necrosis in cochlear hair cells.

Tetrabromodiphenyl ether (BDE-47) is widely used as commercial flame retardants that can be released into the environment and finally enter human body through the food chain. It has been identified to generate neurotoxicity, but little is known about auditory damage and the underlying mechanism following BDE-47 exposure. This study aimed to assess the cell viability with BDE-47 concentration ranging from 0 to 150 µM in mouse organ of Corti-derived cell lines (HEI-OC1). Aryl hydrocarbon receptor (AhR) as an environmental sensor, reactive oxygen species (ROS), NLRP3 inflammasome and p38 MAPK pathways were detected. Results: (1) BDE-47 inhibited the viability in a time- and dose-dependent way in HEI-OC1 cells. Cell cycle was arrested in G1 phase by BDE-47; (2) Elevated intracellular ROS, LDH levels and necrosis were found, which was alleviated by pretreatment with ROS scavenger N-acetylcysteine (NAC); (3) AhR plays an essential role in ligand-regulated transcription factor activation by exogenous environmental compounds. We found increased expression of AhR and decreased downstream targets of CYP 1A1 and CYP 1B1 in BDE-47-treated HEI-OC1 cells, which was reversed by the AhR antagonist CH-223191 for 2 h before BDE-47 exposure. No significant change was detected in CYP 2B; (4) Enhanced expressions of NLRP3 and caspase-1 were induced by BDE-47, with up-regulations of both pro-inflammatory factors for IL-1ß, IL-6 and TNF-α, and anti-inflammatory factors for IL-4, IL-10 and IL-13, but down-regulation for IL-1α; (5) Additionally, the p38 MAPK signaling pathway was activated with increased phosphorylation levels of MKK/3/6, p38 MAPK and NF-kB. Overall, our findings illustrate a role of AhR in ROS-induced necrosis of cochlear hair cells by BDE-47 exposure, in which NLRP3 inflammasome and p38 MAPK signaling pathways are activated. The current study first elucidates the sense of hearing damage induced by BDE-47, and cell-specific or mixture exposures in vivo or human studies are needed to confirm this association.

Salvianolic acid B inhibits ototoxic drug-induced ototoxicity by suppression of the mitochondrial apoptosis pathway.

It has been claimed that salvianolic acid B (Sal B), a natural bioactive antioxidant, exerts protective effects in various types of cells. This study aims to evaluate the antioxidant and anti-apoptosis effects of Sal B in a cultured HEI-OC1 cell line and in transgenic zebrafish (Brn3C: EGFP). A CCK-8 assay, Annexin V Apoptosis Detection Kit, TUNEL and caspase-3/7 staining, respectively, examined apoptosis and cell viability. The levels of reactive oxygen species (ROS) were evaluated by CellROX and MitoSOX Red staining. JC-1 staining was employed to detect the mitochondrial membrane potential (ΔΨm). Western blotting was used to assess expressions of Bax and Bcl-2. The expression pattern of p-PI3K and p-Akt was determined by immunofluorescent staining. We found that Sal B protected against neomycin- and cisplatin-induced apoptotic features, enhanced cell viability and accompanied with decreased caspase-3 activity in the HEI-OC1 cells. Supplementary experiments determined that Sal B reduced ROS production (increased ΔΨm), promoted Bcl-2 expression and down-regulated the expression of Bax, as well as activated PI3K/AKT signalling pathways in neomycin- and cisplatin-injured HEI-OC1 cells. Moreover, Sal B markedly decreased the TUNEL signal and protected against neomycin- and cisplatin-induced neuromast HC loss in the transgenic zebrafish. These results unravel a novel role for Sal B as an otoprotective agent against ototoxic drug-induced HC apoptosis, offering a potential use in the treatment of hearing loss.

Impaired Glycolysis Promotes AlcoholExposure-Induced Apoptosis in HEI-OC1 Cells via Inhibition of EGFR Signaling.

Glucose metabolism is an important metabolic pathway in the auditory system. Chronic alcohol exposure can cause metabolic dysfunction in auditory cells during hearing loss. While alcohol exposure has been linked to hearing loss, the mechanism by which impaired glycolysis promotes cytotoxicity and cell death in auditory cells remains unclear. Here, we show that the inhibition of epidermal growth factor receptor (EGFR)-induced glycolysis is a critical mechanism for alcohol exposure-induced apoptosis in HEI-OC1 cells. The cytotoxicity via apoptosis was significantly increased by alcohol exposure in HEI-OC1 cells. The glycolytic activity and the levels of hexokinase 1 (HK1) were significantly suppressed by alcohol exposure in HEI-OC1 cells. Mechanistic studies showed that the levels of EGFR and AKT phosphorylation were reduced by alcohol exposure in HEI-OC1 cells. Notably, HK1 expression and glycolytic activity was suppressed by EGFR inhibition in HEI-OC1 cells. These results suggest that impaired glycolysis promotes alcohol exposure-induced apoptosis in HEI-OC1 cells via the inhibition of EGFR signaling.

The effect of AP-2δ on transcription of the Prestin gene in HEI-OC1 cells upon oxidative stress.

BACKGROUND: The study aimed to investigate the effect of oxidative stress on Prestin expression, and explore the transcription factors (TFs) that are involved in regulating the expression of Prestin in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells upon oxidative stress. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to detect the expression level of Prestin. Reverse chromatin immunoprecipitation (reverse ChIP) assay was performed to identify proteins that could bind to the Prestin gene. Small interfering RNA (siRNA) and chromatin immunoprecipitation (ChIP) experiments were used to further verify the results. HEI-OC1 cells were incubated with four different concentrations of tert-butyl hydroperoxide (t-BHP) for 24 h or 48 h to construct the oxidative stress model. RESULTS: Oxidative stress induced Prestin increase at the mRNA level but with a concomitant decrease at the protein level. TF activating enhancer binding protein-2δ (AP-2δ) screened by reverse ChIP assay was demonstrated to bind to transcriptional start site 1441 of the Prestin promoter region and negatively regulate the expression of Prestin by siRNA and ChIP experiments. Furthermore, AP-2δ was down-regulated under oxidative stress. CONCLUSIONS: In conclusion, oxidative stress inhibits the expression of Prestin protein, and the transcription mechanism is triggered to compensate for the loss of Prestin protein. AP-2δ is one of the important TFs that suppresses transcription of the Prestin gene, and AP-2δ suppression further boosted Prestin mRNA activation under oxidative stress.

20(S)-Ginsenoside Rh1 inhibits cisplatin-induced hearing loss by inhibiting the MAPK signaling pathway and suppressing apoptosis in vitro.

As an anticancer drug, cisplatin is widely used, but its clinical application is restricted due to its severe side effects of ototoxicity. Therefore, this study was dedicated to assessing the benefit of ginsenoside extract, 20(S)-Ginsenoside Rh1 (Rh1), on cisplatin-induced ototoxicity. HEI-OC1 cells and neonatal cochlear explants were cultured. Cleaved caspase-3, TUNEL, and MitoSOX Red were observed in vitro by immunofluorescence staining. CCK8 and LDH cytotoxicity assays were detected to measure cell viability and cytotoxicity. Our results showed that Rh1 significantly increased cell viability, reduced cytotoxicity, and alleviated cisplatin-induced apoptosis. In addition, Rh1 pretreatment decreased the excessive accumulation of intracellular reactive oxygen species. Mechanistic studies indicated that Rh1 pretreatment reversed the increase of apoptotic protein expression, accumulation of mitochondrial ROS, and activation of the MAPK signaling pathway. These results suggested that Rh1 can act as an antioxidant and anti-apoptotic agent against cisplatin-induced hearing loss by suppressing the excessive accumulation of mitochondrial ROS, activation of MAPK signaling pathway and apoptosis.

Poly ADP-Ribose Polymerase-1 inhibition by 3-aminobenzamide recuperates HEI-OC1 auditory hair cells from blast overpressure-induced cell death.

Introduction: Poly ADP-Ribose Polymerase-1 (PARP1), a DNA repair enzyme is implicated as a key molecule in the pathogenesis of several neurodegenerative disorders. Traumatic insults inducing oxidative stress results in its over-activation causing inflammation and cell death (Parthanatos). As PARP1 inhibition is known to reduce oxidative stress, we hypothesized that PARP1 inhibition by a known inhibitor 3-aminobenzamide (3AB) might recuperate the damage in an in vitro model of blast injury using HEI-OC1 cells (mouse auditory hair cells). Methods: Here, we evaluated the protective effect of 3AB on HEI-OC1 cells following single and repetitive blast overpressures (BOPs). Results: We found that inhibition of PARP1 b 3AB inhibits the PARP1 enzyme and its action of a post-translational modification i.e. formation of Poly ADP-Ribose Polymers which leads to massive ATP depletion. PARP inhibition (3AB treatment) reduced the oxidative stress (4HNE, a marker of lipid peroxidation, and 8OHdG, a marker of oxidative DNA damage) in cells exposed to single/repetitive BOPS through up-regulation of Nrf2, a transcriptional regulator of antioxidant defense and the GCLC, a rate limiting enzyme in the synthesis of glutathione. Discussion: Overall, we found that PARP inhibition by 3AB helps to maintain the viability of BOP-exposed auditory hair cells by recuperating the ATP pool from both mitochondrial and glycolytic sources.

PIN1 protects auditory hair cells from senescence via autophagy.

Background: Age-related hearing loss is an increasing sensorineural hearing loss. But the pathogenesis of ARHL has not been clarified. Herein, we studied the role and significance of PIN1 in regulating autophagy activity in senescence HEI-OC1cells and HCs. Methods and Results: C57BL/6 mice and HEI-OC1 cells were contained in our research. Transfection of plasmids and juglone were used to upregulate or inhibit the PIN 1 expression. Immunofluorescence and Western blot were used to detect the expression of PIN1, LC3, p62, p21 and p16 protein levels in the hair cells of C57BL/6 mice cochleae and HEI-OC1 cells. Senescence-associated ß-galactosidase (SA-ß-gal) staining was used to investigate the senescent level.The results of this study showed that the level of autophagy increased in the senescent auditory hair cells. When inhibited the autophagy level with 3-MA, the senescent HEI-OC1 cells were alleviated. The autophagy activity in senescent HEI-OC1 cells also could be reduced by overexpressing PIN1 protein. On the contrary, inhibiting PIN1 could increase the autophagy level of senescent cells and cochlear hair cells. Conclusion: PIN1 might regulate autophagy activity to induce the senescent of HEI-OC1cells and HCs, which will provide a theoretical support for the prevention and treatment of age-related hearing loss.

MicroRNA Signature and Cellular Characterization of Undifferentiated and Differentiated House Ear Institute-Organ of Corti 1 (HEI-OC1) Cells.

MicroRNAs (miRNAs) regulate gene expressions and control a wide variety of cellular functions. House Ear Institute-Organ of Corti 1 (HEI-OC1) cells are widely used to screen ototoxic drugs and to investigate cellular and genetic alterations in response to various conditions. HEI-OC1 cells are almost exclusively studied under permissive conditions that promote cell replication at the expense of differentiation. Many researchers suggest that permissive culture condition findings are relevant to understanding human hearing disorders. The mature human cochlea however consists of differentiated cells and lacks proliferative capacity. This study therefore aimed to compare the miRNA profiles and cellular characteristics of HEI-OC1 cells cultured under permissive (P-HEI-OC1) and non-permissive (NP-HEI-OC1) conditions. A significant increase in the level of expression of tubulin ß1 class VI (Tubb1), e-cadherin (Cdh1), espin (Espn), and SRY (sex determining region Y)-box2 (Sox2) mRNAs was identified in non-permissive cells compared with permissive cells (P < 0.05, Kruskal-Wallis H test, 2-sided). miR-200 family, miR-34b/c, and miR-449a/b functionally related cluster miRNAs, rodent-specific maternally imprinted gene Sfmbt2 intron 10th cluster miRNAs (-466a/ -467a), and miR-17 family were significantly (P < 0.05, Welch's t-test, 2-tailed) differentially expressed in non-permissive cells when compared with permissive cells. Putative target genes were significantly predominantly enriched in mitogen-activated protein kinase (MAPK), epidermal growth factor family of receptor tyrosine kinases (ErbB), and Ras signaling pathways in non-permissive cells compared with permissive cells. This distinct miRNA signature of differentiated HEI-OC1 cells could help in understanding miRNA-mediated cellular responses in the adult cochlea.

Effects of L-Serine Against Cisplatin-Mediated Reactive Oxygen Species Generation in Zebrafish Vestibular Tissue Culture and HEI-OC1 Auditory Hybridoma Cells.

Cisplatin is a platinum-based chemotherapy compound effective against a variety of cancers. However, it can cause increased reactive oxygen species (ROS) production in auditory and vestibular tissue leading to permanent hearing and balance loss. The amino acid, L-serine, has been shown to reduce ROS in some tissue types. In this project, we first investigated whether L-serine could reduce cisplatin-mediated ROS generation in zebrafish utricular tissue culture using spectrophotometry and the fluorescent ROS detector dye, H2DCFDA. Then, we examined whether L-serine could prevent the effect of cisplatin against cellular viability in the mouse auditory hybridoma cell line, HEI-OC1, using the spectrophotometric (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay. As a final step, we used H2DCFDA dye and flow cytometry analysis to determine if L-serine could counteract the effect of cisplatin on ROS production in this cell line. We found that cisplatin and L-serine treatment may influence ROS production in utricular tissue. Further, although L-serine did not counteract the effect of cisplatin against HEI-OC1 cellular viability, the amino acid did prevent the platinum compound's effect to increase ROS in these cells. These results suggest that L-serine may act in auditory and vestibular tissues as an effective protectant against cisplatin-mediated toxicity.

The monofunctional platinum(II) compounds, phenanthriplatin and pyriplatin, modulate apoptosis signaling pathways in HEI-OC1 auditory hybridoma cells.

Cisplatin is a platinum(II) chemotherapy drug that can cause the side-effect of ototoxicity and hearing loss. The monofunctional platinum(II) complexes, phenanthriplatin and pyriplatin, have recently been investigated as anti-cancer agents but their side-effects are largely unknown. Here, we used the auditory hybridoma cell line, HEI-OC1, to investigate the ototoxicity of cisplatin, phenanthriplatin and pyriplatin. The effect of these compounds against cellular viability, on reactive oxygen species (ROS) production, mitochondrial membrane polarization, caspase-3/7 activity, DNA integrity and caspase-12 expression were measured using spectrophotometric, flow cytometric and blot analyses. We found that the monofunctional complexes and cisplatin decreased cellular viability. All three compounds increased ROS yield at 24 h, but at 48 h, ROS levels returned to normal. Also, the compounds did not depolarize the mitochondrial membrane. All three compounds reduced caspase-3/7 activity at 24 h; cisplatin increased caspase-3/7 activity and caused apoptosis at 48 h. Caspase-12 expression was associated with all three compounds. In summary, the monofunctional complexes may cause ototoxicity like cisplatin. Phenanthriplatin and pyriplatin may cause ototoxicity initially by inducing ROS production, but they may also signal through distinct apoptotic pathways that do not integrate caspases-3/7, or may act at different time-points in the same pathways.

Preliminary Characterization of Extracellular Vesicles From Auditory HEI-OC1 Cells.

OBJECTIVES: Isolate, purify, and characterize extracellular vesicles (EVs) obtained from auditory HEI-OC1 cells, and evaluate their suitability for intracochlear transport and delivery of pharmacological drugs and/or pro-resolution mediators of acute inflammatory processes. METHODS: HEI-OC1 EVs were isolated and purified using the exoEasy Maxi Kit, and their size was evaluated by nanoparticle tracking techniques. Bottom-up proteomics of the EVs, either freshly obtained or stored for up to 4 months at -20°C, was performed by LC-ESI-MS/MS. LC-ESI-MS/MS-MRM was used to measure the loading of dexamethasone inside EVs following co-incubation at room temperature for 1 hour with and without 5 minutes sonication. RESULTS: Routinely, we were able to obtain purified fractions of >2 × 109 EVs/mL, with diameters varying between 50 and 800 nm. Bottom-up proteomics showed that among the most abundant EVs proteins, 19.2% were cytoplasmic, 17.2% were membrane localized, 12.3% were cytosolic, and 14.6% were nucleolar. No significant differences between fresh and stored EVs were detected. Importantly, co-incubation of HEI-OC1 EVs (1 × 108 EVs/mL) with dexamethasone (10 mM) resulted in the incorporation of 10.1 ± 1.9 nM dexamethasone per milliliter of EVs suspension. CONCLUSIONS: Altogether, the results suggest that EVs from HEI-OC1 cells could be advantageously used as biological nanocarriers for the delivery of specific molecules and pharmacological drugs into the inner ear.

Fetal bovine serum inhibits neomycin-induced apoptosis of hair cell-like HEI-OC-1 cells by maintaining mitochondrial function.

Aging and exposure to noise or ototoxic drugs are major causes of hair cell death leading to human hearing loss, and many agents have been developed to protect hair cells from apoptosis. Fetal bovine serum (FBS) is a fundamental ingredient in the culture medium of hair cell-like House Ear Institute Organ of Corti 1 (HEI-OC-1) cells, but there have been no reports about the function of FBS in HEI-OC-1 cell apoptosis. In this study, we found that FBS deprivation alone significantly increased HEI-OC-1 cell apoptosis in the absence of neomycin exposure and that the presence of FBS significantly inhibited HEI-OC-1 cell apoptosis after neomycin exposure compared to FBS-deprived cells. Further, we found that the protective effect of FBS was dose dependent and more effective than the growth factors B27, N2, EGF, bFGF, IGF-1, and heparan sulfate. We also found that FBS deprivation significantly disrupted the expression level of mitochondrial proteins, increased pro-apoptotic gene expression, decreased the mitochondrial membrane potential, and increased reactive oxygen species accumulation in HEI-OC-1 cells after neomycin exposure. These findings indicate that FBS is involved in maintaining the level of mitochondrial proteins, maintaining the balance of oxidant gene expression, and preventing the accumulation of ROS, and thus FBS maintains normal mitochondrial function and inhibits apoptosis in HEI-OC-1 cells after neomycin exposure.

Extracellular Vesicles From Auditory Cells as Nanocarriers for Anti-inflammatory Drugs and Pro-resolving Mediators.

Drug- and noise-related hearing loss are both associated with inflammatory responses in the inner ear. We propose that intracochlear delivery of a combination of pro-resolving mediators, specialized proteins and lipids that accelerate the return to homeostasis by modifying the immune response rather than by inhibiting inflammation, might have a profound effect on the prevention of sensorineural hearing loss. However, intracochlear delivery of such agents requires a reliable and effective method to convey them, fully active, directly to the target cells. The present study provides evidence that extracellular vesicles (EVs) from auditory HEI-OC1 cells may incorporate significant quantities of anti-inflammatory drugs, pro-resolving mediators and their polyunsaturated fatty acid precursors as cargo, and potentially could work as carriers for their intracochlear delivery. EVs generated by HEI-OC1 cells were divided by size into two fractions, small (≤150 nm diameter) and large (>150 nm diameter), and loaded with aspirin, lipoxin A4, resolvin D1, and the polyunsaturated fatty acids (PUFA) arachidonic, eicosapentaenoic, docosahexanoic, and linoleic. Bottom-up proteomics revealed a differential distribution of selected proteins between small and large vesicles. Only 17.4% of these proteins were present in both fractions, whereas 61.5% were unique to smaller vesicles and only 3.7% were exclusively found in the larger ones. Importantly, the pro-resolving protein mediators Annexin A1 and Galectins 1 and 3 were only detected in small vesicles. Lipidomic studies, on the other hand, showed that small vesicles contained higher levels of eicosanoids than large ones and, although all of them incorporated the drugs and molecules investigated, small vesicles were more efficiently loaded with PUFA and the large ones with aspirin, LXA4 and resolvin D1. Importantly, our data indicate that the vesicles contain all necessary enzymatic components for the de novo generation of eicosanoids from fatty acid precursors, including pro-inflammatory agents, suggesting that their cargo should be carefully tailored to avoid interference with their therapeutic purpose. Altogether, these results support the idea that both small and large EVs from auditory HEI-OC1 cells could be used as nanocarriers for anti-inflammatory drugs and pro-resolving mediators.

Meclofenamic Acid Reduces Reactive Oxygen Species Accumulation and Apoptosis, Inhibits Excessive Autophagy, and Protects Hair Cell-Like HEI-OC1 Cells From Cisplatin-Induced Damage.

Hearing loss is the most common sensory disorder in humans, and a significant number of cases is due to the ototoxicity of drugs such as cisplatin that cause hair cell (HC) damage. Thus, there is great interest in finding agents and mechanisms that protect HCs from ototoxic drug damage. It has been proposed that epigenetic modifications are related to inner ear development and play a significant role in HC protection and HC regeneration; however, whether the m6A modification and the ethyl ester form of meclofenamic acid (MA2), which is a highly selective inhibitor of FTO (fatmass and obesity-associated enzyme, one of the primary human demethylases), can affect the process of HC apoptosis induced by ototoxic drugs remains largely unexplored. In this study, we took advantage of the HEI-OC1 cell line, which is a cochlear HC-like cell line, to investigate the role of epigenetic modifications in cisplatin-induced cell death. We found that cisplatin injury caused reactive oxygen species accumulation and increased apoptosis in HEI-OC1 cells, and the cisplatin injury was reduced by co-treatment with MA2 compared to the cisplatin-only group. Further investigation showed that MA2 attenuated cisplatin-induced oxidative stress and apoptosis in HEI-OC1 cells. We next found that the cisplatin-induced upregulation of autophagy was significantly inhibited after MA2 treatment, indicating that MA2 inhibited the cisplatin-induced excessive autophagy. Our findings show that MA2 has a protective effect and improves the viability of HEI-OC1 cells after cisplatin treatment, and they provide new insights into potential therapeutic targets for the amelioration of cisplatin-induced ototoxicity.

Decreased Expression of TRPV4 Channels in HEI-OC1 Cells Induced by High Glucose Is Associated with Hearing Impairment.

PURPOSE: Previous reports have shown that hyperglycemia-induced inhibition of transient receptor potential vanilloid sub type 4 (TRPV4), a transient receptor potential ion channel, affects the severity of hearing impairment (HI). In this study, we explored the role of TRPV4 in HI using HEI-OC1 cells exposed to high glucose (HG). MATERIALS AND METHODS: HEI-OC1 cells were cultured in a HG environment (25 mM D-glucose) for 48 hours, and qRT-PCR and Western blotting were used to analyze the expression of TRPV4 at the mRNA and protein level. TRPV4 agonist (GSK1016790A) or antagonist (HC-067047) in cultured HEI-OC1 cells was used to obtain abnormal TRPV4 expression. Functional TRPV4 activity was assessed in cultured HEI-OC1 cells using the MTT assay and a cell death detection ELISA. RESULTS: TRPV4 agonists exerted protective effects against HG-induced HI, as evidenced by increased MTT levels and inhibition of apoptosis in HEI-OC1 cells. TRPV4 overexpression significantly increased protein levels of phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK), while TRPV4 antagonists had the opposite effect. Our results indicated that TRPV4 is a hyperglycemia-related factor that can inhibit cell proliferation and promote cell apoptosis by activating the MAPK signaling pathway in HEI-OC1 cells. CONCLUSION: Our results show that the overexpression of TRPV4 can attenuate cell death in HEI-OC1 cells exposed to HG.

HEI-OC1 cells as a model for investigating prestin function.

The House Ear Institute-Organ of Corti 1 (HEI-OC1) is a mouse auditory cell line that endogenously express, among other several markers of cochlear hair cells, the motor protein prestin (SLC26A5). Since its discovery fifteen years ago, and because of the difficulties associated with working with outer hair cells, prestin studies have been performed mostly by expressing it exogenously in non-specific systems such as HEK293 and TSA201, embryonic kidney cells from human origin, or Chinese Hamster Ovary (CHO) cells. Here, we report flow cytometry and confocal laser scanning microscopy studies on the pattern of prestin expression, as well as nonlinear capacitance (NLC) and whole cell-patch clamping studies on prestin motor function, in HEI-OC1 cells cultured at permissive and non-permissive conditions. Our results indicate that both total prestin expression and plasma membrane localization increase in a time-dependent manner when HEI-OC1 cells differentiate under non-permissive culture conditions. In addition, we demonstrate that HEI-OC1 cells have a robust NLC associated to prestin motor function, which decreases when the density of prestin molecules present at the plasma membrane increases. Altogether, our results show that the response of endogenously expressed prestin in HEI-OC1 cells is different from the response of prestin expressed exogenously in non-auditory cells, and suggest that the HEI-OC1 cell line may be an important additional tool for investigating prestin function.

HEI-OC1 cells as a model for investigating drug cytotoxicity.

The House Ear Institute-Organ of Corti 1 (HEI-OC1) is one of the few, and arguable the most used, mouse auditory cell line available for research purposes. Originally proposed as an in vitro system for screening of ototoxic drugs, it has been used to investigate, among other topics, apoptotic pathways, autophagy and senescence, mechanism of cell protection, inflammatory responses, cell differentiation, effects of hypoxia, oxidative and endoplasmic reticulum stress, and expression of molecular channels and receptors. However, the use of different techniques with different goals resulted in apparent contradictions on the actual response of these cells to some specific treatments. We have now performed studies to characterize the actual response of HEI-OC1 cells to a battery of commonly used pharmacological drugs. We evaluated cell toxicity, apoptosis, viability, proliferation, senescence and autophagy in response to APAP (acetaminophen), cisplatin, dexamethasone, gentamicin, penicillin, neomycin, streptomycin, and tobramycin, at five different doses and two time-points (24 and 48 h), by flow cytometry techniques and caspase 3/7, MTT, Cytotoxicity, BrdU, Beclin1, LC3 and SA-ß-galactosidase assays. We also used HEK-293 and HeLa cells to compare some of the responses of these cells to those of HEI-OC1. Our results indicate that every cell line responds to the each drug in a different way, with HEI-OC1 cells showing a distinctive sensitivity to at least one of the mechanisms under study. Altogether, our results suggest that the HEI-OC1 might be a useful model to investigate biological responses associated with auditory cells, including auditory sensory cells, but a careful approach would be necessary at the time of evaluating drug effects.

Development of Cell-Based High-Throughput Chemical Screens for Protection Against Cisplatin-Induced Ototoxicity.

Various compounds have been tested in recent years for protection against cisplatin-induced hearing loss, but no compound has yet been FDA approved for clinical use in patients. Towards this goal, we developed an unbiased, high-throughput, mammalian cochlear cell-based chemical screen that allowed quantification of the protection ability of bioactive compounds and ranked them for future testing ex vivo in cochlear explant cultures and in vivo in animal models. In our primary screens, protection in the HEI-OC1 organ of Corti immortalized cell line was measured by the ability of each compound to inhibit caspase-3/7 activity triggered by cisplatin treatment (50 µM cisplatin for 22 h). A total of 4385 unique bioactive compounds were tested in a single dose of 8 µM and promising compounds were validated by dose response curves covering ten, 1:3 serial diluted concentrations. Primary hits were defined as having more than 60 % inhibition of the caspase-3/7 activity. Toxicity of the top compounds was measured by a CellTiter-Glo (CTG) assay that measured the viability of the cells in the presence of compound alone in similar dose responsive analysis. A combination of the caspase-3/7 inhibition activity assay (as measured by IC50) and the CTG viability assay (as determined by LD50) identified the top protective compounds in the HEI-OC1 cells. In the future, the top hits in our screens will be tested for their protective ability ex vivo in mouse cochlear explants and in vivo in animal models. Our mammalian cochlear cell-based, high-throughput chemical screening assays described here can be further modified and represent an initial successful step towards therapeutic intervention of hearing disorders, an unmet medical need of our society.

Immunological role of prostaglandin E2 production in mouse auditory cells in response to LPS.

The effect of LPS on the production of prostaglandin E2 (PGE2) in mouse HEI-OC1 auditory cells was examined. HEI-OC1 auditory cells constitutively produce a small amount of PGE2. LPS augmented the PGE2 production via enhanced cyclooxygenase 2 (COX2) expression. LPS-induced augmentation of COX2 expression was dependent on up-regulation of COX2 mRNA expression. LPS induced the production of TNF-α, but not IL-1ß· An anti-TNF-α neutralizing Ab significantly inhibited PGE2 production and COX2 mRNA expression in response to LPS. LPS-induced PGE2 production was prevented by a series of pharmacological signaling inhibitors to NF-κB and MAPKs. Pam3CSK4 as a TLR2 ligand, as well as LPS as a TLR4 ligand, augmented the PGE2 production. However, poly I:C as a TLR3 ligand, imiquimod as a TLR7 ligand and CpG DNA as a TLR9 ligand did not augment it. HEI-OC1 cells expressed TLR2, TLR4 and TLR9, but not TLR3 or TLR7. The putative role of LPS-induced PGE2 production in auditory cells is discussed.