Advertisement

Effects of salicylate derivatives on localization of p.H723R allele product of SLC26A4

Published:March 16, 2022DOI:https://doi.org/10.1016/j.anl.2022.03.009

      Abstract

      Objective

      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.

      Methods

      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.

      Results

      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.

      Conclusion

      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.

      Keywords

      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'

      Subscribe:

      Subscribe to Auris Nasus Larynx
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect

      References

        • Everett L.A.
        • Glaser B.
        • Beck J.C.
        • Idol J.R.
        • Buchs A.
        • Heyman M.
        • et al.
        Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS).
        Nat Genet. 1997; 17: 411-422
        • Royaux I.E.
        • Suzuki K.
        • Mori A.
        • Katoh R.
        • Everett L.A.
        • Kohn L.D.
        • et al.
        Pendrin, the protein encoded by the Pendred syndrome gene (PDS), is an apical porter of iodide in the thyroid and is regulated by thyroglobulin in FRTL-5 cells.
        Endocrinology. 2000; 141: 839-845
        • Bassot C.
        • Minervini G.
        • Leonardi E.
        • Tosatto SC.
        Mapping pathogenic mutations suggests an innovative structural model for the pendrin (SLC26A4) transmembrane domain.
        Biochimie. 2017; 132: 109-120
        • Kuwabara M.F.
        • Wasano K.
        • Takahashi S.
        • Bodner J.
        • Komori T.
        • Uemura S.
        • et al.
        The extracellular loop of pendrin and prestin modulates their voltage-sensing property.
        J Biol Chem. 2018; 293: 9970-9980
        • Scott D.A.
        • Wang R.
        • Kreman T.M.
        • Sheffield V.C.
        • Karniski LP.
        The Pendred syndrome gene encodes a chloride-iodide transport protein.
        Nat Genet. 1999; 21: 440-443
        • Royaux I.E.
        • Wall S.M.
        • Karniski L.P.
        • Everett L.A.
        • Suzuki K.
        • Knepper M.A.
        • et al.
        Pendrin, encoded by the Pendred syndrome gene, resides in the apical region of renal intercalated cells and mediates bicarbonate secretion.
        Proc Natl Acad Sci USA. 2001; 98: 4221-4226
        • Yoshida A.
        • Taniguchi S.
        • Hisatome I.
        • Royaux I.E.
        • Green E.D.
        • Kohn L.D.
        • et al.
        Pendrin is an iodide-specific apical porter responsible for iodide efflux from thyroid cells.
        J Clin Endocrinol Metab. 2002; 87: 3356-3361
        • Gillam M.P.
        • Sidhaye A.R.
        • Lee E.J.
        • Rutishauser J.
        • Stephan C.W.
        • Kopp P.
        Functional characterization of pendrin in a polarized cell system. Evidence for pendrin-mediated apical iodide efflux.
        J Biol Chem. 2004; 279: 13004-13010
        • Wangemann P.
        • Nakaya K.
        • Wu T.
        • Maganti R.J.
        • Itza E.M.
        • Sanneman J.D.
        • et al.
        Loss of cochlear HCO3 secretion causes deafness via endolymphatic acidification and inhibition of Ca2+ reabsorption in a Pendred syndrome mouse model.
        Am J Physiol Renal Physiol. 2007; 292: F1345-F1353
        • Kopp P.
        • Pesce L.
        • Solis-S J.C.
        Pendred syndrome and iodide transport in the thyroid.
        Trends Endocrinol Metab. 2008; 19: 260-268
        • Mount D.B.
        • Romero MF.
        The SLC26 gene family of multifunctional anion exchangers.
        Pflugers Arch. 2004; 447: 710-721
        • Usami S.
        • Abe S.
        • Weston M.D.
        • Shinkawa H.
        • Van Camp G.
        • Kimberling WJ.
        Non-syndromic hearing loss associated with enlarged vestibular aqueduct is caused by PDS mutations.
        Hum Genet. 1999; 104: 188-192
        • Kopp P.
        Pendred syndrome: clinical characteristics and molecular basis.
        Curr Opin Endocrinol Diabetes. 1999; 6: 261-268
        • Dossena S.
        • Rodighiero S.
        • Vezzoli V.
        • Nofziger C.
        • Salvioni E.
        • Boccazzi M.
        • et al.
        Functional characterization of wild-type and mutated pendrin (SLC26A4), the anion transporter involved in Pendred syndrome.
        J Mol Endocrinol. 2009; 43: 93-103
        • Tsukamoto K.
        • Suzuki H.
        • Harada D.
        • Namba A.
        • Abe S.
        • Usami S.
        Distribution and frequencies of PDS (SLC26A4) mutations in Pendred syndrome and nonsyndromic hearing loss associated with enlarged vestibular aqueduct: a unique spectrum of mutations in Japanese.
        Eur J Hum Genet. 2003; 11: 916-922
        • Miyagawa M.
        • Nishio S.Y.
        • Usami S.
        Mutation spectrum and genotype-phenotype correlation of hearing loss patients caused by SLC26A4 mutations in the Japanese: a large cohort study.
        J Hum Genet. 2014; 59: 262-268
        • Ishihara K.
        • Okuyama S.
        • Kumano S.
        • Iida K.
        • Hamana H.
        • Murakoshi M.
        • et al.
        Salicylate restores transport function and anion exchanger activity of missense pendrin mutations.
        Hear Res. 2010; 270: 110-118
        • Boettcher F.A.
        • Salvi RJ.
        Salicylate ototoxicity: review and synthesis.
        Am J Otolaryngol. 1991; 12: 33-47
        • Kanda Y.
        Investigation of the freely available easy-to-use software ‘EZR’ for medical statistics.
        Bone Marrow Transplant. 2013; 48: 452-458
        • Hosoya M.
        • Fujioka M.
        • Sone T.
        • Okamoto S.
        • Akamatsu W.
        • Ukai H.
        • et al.
        Cochlear cell modeling using disease-specific iPSCs unveils a degenerative phenotype and suggests treatments for congenital progressive hearing loss.
        Cell Rep. 2017; 18: 68-81
        • Hosoya M.
        • Saeki T.
        • Saegusa C.
        • Matsunaga T.
        • Okano H.
        • Fujioka M.
        • et al.
        Estimating the concentration of therapeutic range using disease-specific iPS cells: low-dose rapamycin therapy for Pendred syndrome.
        Regen Ther. 2019; 10: 54-63
        • Rotman-Pikielny P.
        • Hirschberg K.
        • Maruvada P.
        • Suzuki K.
        • Royaux I.E.
        • Green E.D.
        • et al.
        Retention of pendrin in the endoplasmic reticulum is a major mechanism for Pendred syndrome.
        Hum Mol Genet. 2002; 11: 2625-2633
        • Yoon J.S.
        • Park H.J.
        • Yoo S.Y.
        • Namkung W.
        • Jo M.J.
        • Koo S.K.
        • et al.
        Heterogeneity in the processing defect of SLC26A4 mutants.
        J Med Genet. 2008; 45: 411-419
        • Zheng J.
        • Shen W.
        • He D.Z.
        • Long K.B.
        • Madison L.D.
        • Dallos P.
        Prestin is the motor protein of cochlear outer hair cells.
        Nature. 2000; 405: 149-155
        • Dallos P.
        • Fakler B.
        Prestin, a new type of motor protein.
        Nat Rev Mol Cell Biol. 2002; 3: 104-111
        • Kumano S.
        • Iida K.
        • Ishihara K.
        • Murakoshi M.
        • Tsumoto K.
        • Ikeda K.
        • et al.
        Salicylate-induced translocation of prestin having mutation in the GTSRH sequence to the plasma membrane.
        FEBS Lett. 2010; 584: 2327-2332
        • Kalinec G.M.
        • Webster P.
        • Lim D.J.
        • Kalinec F.
        A cochlear cell line as an in vitro system for drug ototoxicity screening.
        Audiol Neurootol. 2003; 8: 177-189
        • Kalinec G.
        • Thein P.
        • Park C.
        • Kalinec F.
        HEI-OC1 cells as a model for investigating drug cytotoxicity.
        Hear Res. 2016; 335: 105-117
        • Taubes G.
        Misfolding the way to disease.
        Science. 1996; 271: 1493-1495
        • Welch W.J.
        • Brown CR.
        Influence of molecular and chemical chaperones on protein folding.
        Cell Stress Chaperones. 1996; 1: 109-115
        • Soto C.
        Protein misfolding and disease; protein refolding and therapy.
        FEBS Lett. 2001; 498: 204-207
        • Cohen F.E.
        • Kelly JW.
        Therapeutic approaches to protein-misfolding diseases.
        Nature. 2003; 426: 905-909
        • Conn P.M.
        • Janovick JA.
        A new understanding of protein mutation unfolds.
        Am Sci. 2005; 93: 314-321
        • Conn P.M.
        • Ulloa-Aguirre A.
        Pharmacological chaperones for misfolded gonadotropin-releasing hormone receptors.
        Adv Pharmacol. 2011; 62: 109-141
        • Janovick J.A.
        • Maya-Nunez G.
        • Conn PM.
        Rescue of hypogonadotropic hypogonadism-causing and manufactured GnRH receptor mutants by a specific protein-folding template: misrouted proteins as a novel disease etiology and therapeutic target.
        J Clin Endocrinol Metab. 2002; 87: 3255-3262