SERCA2 protects against cisplatin-induced damage of auditory cells: Possible relation with alleviation of ER stress.

AIMS: SERCA2, one of the P-type pumps encoded by gene ATP2A2, is the only calcium reflux channel of the endoplasmic reticulum (ER) and participates in maintaining calcium homeostasis. The present study was designed to explore SERCA2 expression pattern in auditory hair cells and the possible mechanism underlying the effects of SERCA2 on cisplatin-induced ototoxicity. MAIN METHODS: The SERCA2 expression pattern in cochlea hair cells and HEI-OC1 cells was measured by Western blot (WB) and immunofluorescence staining. The apoptosis and its related factors were detected by TUNEL assay and WB. The expression levels of ER stress-related factors, ATF6, PERK, IRE1α, and GRP78, were measured via WB. As for the determination of SERCA2 overexpression and knockdown, plasmids and lentiviral vectors were constructed, respectively. KEY FINDINGS: We found that SERCA2 was highly expressed in cochlea hair cells and HEI-OC1 cells. Of note, the level of SERCA2 expression in neonatal mice was remarkably higher than that in adult mice. Under the exposure of 30 µM cisplatin, SERCA2 was down-regulated significantly compared with the control group. In addition, cisplatin administration triggered the occurrence of ER stress and apoptosis. Those events were reversed by overexpressing SERCA2. On the contrary, SERCA2 knockdown could aggravate the above processes. SIGNIFICANCE: The findings from the present study disclose, for the first time, that SERCA2 is abundantly expressed in cochlea hair cells, and the suppression of SERCA2 caused by cisplatin could trigger ER homeostasis disruption, thereby implying that SERCA2 might be a promising target to prevent cisplatin-induced cytotoxicity of hair cells.

AdipoRon reduces cisplatin-induced ototoxicity in hair cells:possible relation to the regulation of mitochondrial biogenesis.

AdipoRon (AR) can exert antidiabetic and anti-inflammatory effects by maintaining mitochondrial structure and function. The present study was designed to explore whether AR protects the auditory cells from cisplatin-induced damage and, if so, to probe the possible mechanisms underlying its action on this type of cells. Cell viability and apoptosis in House Ear Institute-Organization of Corti 1 (HEI-OC1 cells) and mouse cochlea hair cells (HCs) were detected by CCK8 and immunofluorescence. The expressions of apoptosis-related proteins (cleaved caspase-3 and Bcl-2), adiponectin receptor 1 (AdipoR 1) and the key factors relevant to mitochondrial biogenesis（SIRT1 and TFAM）were determined by Western blot and immunofluorescence. Changes in apoptotic rate and expression of SIRT1 and TFAM after silencing of AdipoR 1 (AdipoR 1-siRNA) in HEI-OC1 cells were measured by flow cytometry and Western blot. The levels of reactive oxygen species (ROS) were evaluated by MitoSox red staining. We found that 30 µM cisplatin exposure induced severe cellular damage, which resulted from activation of the mitochondrial apoptotic pathway. Cisplatin decreased the expression of AdipoR 1, SIRT1, and TFAM proteins, leading to impaired mitochondrial biogenesis and increased mitochondrial ROS production. 10 µM AR pre-treatment enhanced mitochondrial biogenesis, decreased mitochondrial ROS levels, alleviated imbalances in the mitochondrial apoptotic pathway, thus reducing cisplatin-induced apoptosis. Taken together, this work reveals that AR exerts anti-apoptotic effects, possibly via regulating mitochondrial biogenesis and function. Interestingly, AR might possess the promising potential to be a novel drug for the prevention and/ or treatment of cisplatin-induced ototoxicity.

3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR) protects hair cells from cisplatin-induced ototoxicity in vitro: possible relation to the activities of p38 MAPK signaling pathway.

The 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR) gene encodes rate-limiting enzyme in cholesterol biosynthesis, which is related to cell proliferation and mitochondrial function. The present study was designed to explore the expression of HMGCR in murine cochlear hair cells and HEI-OC1 cells and the possible mechanisms underpinning the actions of HMGCR in cisplatin-induced ototoxicity, with special attention given to p38 mitogen-activated protein kinase (MAPK) activities in vitro. The expressions of HMGCR, p-p38, cleaved caspase-3 and LC3B was measured by immunofluorescence and western blot. JC-1 staining and MitoSOX Red were used to detect mitochondria membrane potential (MMP) and reactive oxygen species (ROS) levels respectively. The apoptosis of auditory cells was assessed by TUNEL staining and flow cytometry. Protein levels of bcl2/bax and beclin1 were examined by western blot. We found that HMGCR was widely expressed in the auditory cells, of both neonatal mice and 2-month-old mice, in cytoplasm, nucleus and stereocilia. Moreover, 30 µM cisplatin elicited the formation of ROS, which, in turn, led to HMGCR reduction, activating p38 kinase-related apoptosis and autophagy in auditory cells. Meanwhile, co-treatment with ROS scavenger at a concentration of 2 mM, N-acetyl-L-cysteine (NAC), could alleviate the aforementioned changes. In addition, HMGCR silencing resulted in higher p38 MAPK-mediated apoptosis and autophagy under cisplatin injury. Taken together, we demonstrate that, for the first time, that HMGCR is expressed in the cochlear. Furthermore, HMGCR exerts protective benefit on auditory cells against cisplatin-mediated injury stimulated by ROS, culminating in regulation of p38 MAPK-dependent apoptosis and autophagy.

DJ-1 Protects auditory cells from cisplatin-induced ototoxicity via regulating apoptosis and autophagy.

AIMS: DJ-1, a multifunctional protein encoded by the Park7 gene, is tightly related to mitochondrial dysfunction, oxidative stress, protein aggregation, and autophagy regulation. The current study was designed to investigate whether DJ-1 is expressed in auditory cells and, if so, to explore the possible correlation between DJ-1 and cisplatin-induced ototoxicity in this type of cells. METHODS: The location and dynamic expression of DJ-1 in mouse cochlea hair cells (HCs) and House Ear Institute-Organ of Corti 1 (HEI-OC1 cells) were detected by immunofluorescence, real-time PCR, and western blot. The apoptosis of auditory cells was assessed by TUNEL staining and flow cytometry. The levels of ROS were evaluated by MitoSox red staining. The expression of protein cleaved caspase-9, cleaved caspase-3, and LC3B was examined by immunofluorescence and western blot. The expressions of certain key factors relevant to apoptosis (Bcl-2 and Bax) and autophagy (Beclin1, p-JNK, and p-c-Jun) were determined by western blot. The dynamic alterations of those factors in response to DJ-1 knockdown in HEI-OC1 cells (DJ-1-KD) were measured by western blot and MitoSox red staining. RESULTS: The expression of DJ-1 was clearly shown in both HCs and HEI-OC1 cells and cisplatin led to the reduction of DJ-1 expression in a concentration and time-dependent manner. Meanwhile, cisplatin-induced apoptotic process was implemented by promoting reactive oxygen species (ROS) production and activating the mitochondrial pathway. Furthermore, DJ-1 explicitly participated in cisplatin-trigged cell damage by regulating autophagy. CONCLUSIONS: Findings from this work clearly reveal, for the first time, that DJ-1 is expressed in the cochlea. Of particular importance, DJ-1 exerts its protective action against cisplatin-elicited injury on auditory cells via regulating apoptosis and autophagy, which provides a new strategy for the prevention of cisplatin-induced ototoxicity.