Faculty of Frontier Engineering, Institute of Science and Engineering, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan; Division of Mechanical Science and Engineering, Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa, Japan.
Division of Mechanical Science and Engineering, Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa, Japan.
Auris Nasus Larynx. 2022 Dec;49(6):928-937. doi: 10.1016/j.anl.2022.03.009. Epub 2022 Mar 16.
Pendrin is a transmembrane protein encoded by the SLC26A4 gene that functions in maintaining ion concentrations in the endolymph of the inner ear, most likely by acting as a chloride/bicarbonate transporter. Variants in the SLC26A4 gene are responsible for sensorineural hearing loss. Although pendrin localizes to the plasma membrane, we previously identified that 8 missense allele products of SLC26A4 were retained in the intracellular region and lost their anion exchange function. We also found that 10 mM salicylate induced the translocation of 4 out of 8 allele products from the intracellular region to the plasma membrane and restored their anion exchanger activity. However, since 10 mM salicylate exhibits cytotoxicity, the use of chemical compounds with less cell toxicity is needed. In the present study, therefore, salicylate derivatives were used as the chemical compounds and their effects on the p.H723R allele products of SLC26A4 were investigated.
HEK293 cells were transfected with the cDNA of p.H723R. Cell proliferation, viability and toxicity assays were performed to investigate the response and health of cells in culture after treatment with four types of salicylate derivatives, i.e., 2-hydroxybenzyl alcohol, 2,3-dihydroxybenzoic acid, 2'-hydroxyacetophenone and methyl salicylate. The effects of these salicylate derivatives on the localization of the p.H723R were investigated by immunofluorescence microscopy.
The application of 10 mM salicylate showed an increase in cell toxicity and decrease in cell viability, leading to a significant decrease in cell proliferation. In contrast, the application of 1 mM salicylate derivatives did not show any significant increase in cell toxicity and decrease in cell viability, corresponding to a logarithmic increase in cell concentration with an increase in culture time. Immunofluorescence experiments showed that the p.H723R retained in the endoplasmic reticulum (ER). Among the salicylate derivatives applied, 2-hydroxybenzyl alcohol induced the translocation of p.H723R from the ER to the plasma membrane 3 h after its application.
The results obtained showed that 2-hydroxybenzyl alcohol restored the localization of the p.H723R allele products of SLC26A4 from the ER to the plasma membrane at a concentration of 1 mM by 3 h after its administration with less cytotoxicity than 10 mM salicylate.
Pendrin 是一种跨膜蛋白，由 SLC26A4 基因编码，其功能是维持内耳内淋巴中的离子浓度，可能通过充当氯离子/碳酸氢根转运体起作用。SLC26A4 基因的变异导致感觉神经性听力损失。尽管 pendrin 定位于质膜，但我们之前已经确定 SLC26A4 的 8 种错义等位基因产物在内质网中保留，并丧失了阴离子交换功能。我们还发现，10mM 水杨酸盐诱导 8 种等位基因产物中的 4 种从内质网转移到质膜，并恢复了它们的阴离子交换活性。然而，由于 10mM 水杨酸盐具有细胞毒性，因此需要使用细胞毒性较小的化学化合物。因此，在本研究中，我们使用水杨酸盐衍生物作为化学化合物，并研究它们对 SLC26A4 的 p.H723R 等位基因产物的影响。
用 p.H723R 的 cDNA 转染 HEK293 细胞。通过细胞增殖、活力和毒性测定来研究用四种水杨酸盐衍生物（2-羟基苯甲醇、2,3-二羟基苯甲酸、2'-羟基苯乙酮和甲基水杨酸）处理后培养细胞的反应和健康状况。通过免疫荧光显微镜研究了这些水杨酸盐衍生物对 p.H723R 定位的影响。
应用 10mM 水杨酸盐会增加细胞毒性并降低细胞活力，从而导致细胞增殖显著减少。相比之下，应用 1mM 水杨酸盐衍生物不会导致细胞毒性明显增加和细胞活力降低，对应于细胞浓度随培养时间的对数增加。免疫荧光实验表明，p.H723R 保留在内质网（ER）中。在所应用的水杨酸盐衍生物中，2-羟基苯甲醇在应用后 3 小时诱导 p.H723R 从内质网转位到质膜。
研究结果表明，2-羟基苯甲醇以 1mM 的浓度在给药后 3 小时将 SLC26A4 的 p.H723R 等位基因产物从 ER 恢复到质膜的定位，其细胞毒性小于 10mM 水杨酸盐。
