Osbpl2 deficiency inhibits Rho/ROCK2/p-ERM signaling and impairs actin cytoskeletal regulation in auditory cells

Abstract

A mutation in oxysterol-binding protein-like 2 (OSBPL2) has been identified as the genetic cause of autosomal dominant nonsyndromic hearing loss (DFNA67, Online Mendelian Inheritance in Man No. 616340). However, the pathogenesis of the OSBPL2 mutation in DFNA remains unclear. Our previous work showed that Osbpl2 deficiency impaired cell adhesion in auditory HEI-OC1 cells. In addition, loss of hair cells (HCs) and morphological abnormalities of HC stereocilia were detected in OSBPL2-knockout pigs, suggesting that OSBPL2 plays an important role in regulating the actin cytoskeleton in auditory cells. In the present study, we found that Osbpl2 deficiency inhibited the Rho/ROCK2 signaling pathway and downregulated phosphorylated ezrin-radixin-moesin (p-ERM), resulting in abnormal F-actin morphology in HEI-OC1 cells and stereociliary defects in mouse HCs. The present study demonstrates the underlying mechanism of OSBPL2 in regulating the actin cytoskeleton in HCs, contributing to a deeper understanding of the pathogenesis of OSBPL2 mutations in DFNA.

Keywords

OSBPL2 / hearing loss / actin cytoskeleton / Rho/ROCK signaling / p-ERM

Fundings

This work was financially supported by the National Natural Science Foundation of China (Grant Nos. 82071052 to J.Y. and 81771000 to X.C.).

Acknowledgments

None.

References

[1] Sawamura S, Ogata G, Asai K, et al. Analysis of pharmacokinetics in the cochlea of the inner ear[J]. Front Pharmacol, 2021, 12: 633505. doi: 10.3389/fphar.2021.633505

[2] Kraatari-Tiri M, Haanpää MK, Willberg T, et al. Clinical and genetic characteristics of Finnish patients with autosomal recessive and dominant non-syndromic hearing loss due to pathogenic TMC1 variants[J]. J Clin Med, 2022, 11(7): 1837. doi: 10.3390/jcm11071837

[3] Francis SP, Krey JF, Krystofiak ES, et al. A short splice form of Xin-actin binding repeat containing 2 (XIRP2) lacking the Xin repeats is required for maintenance of stereocilia morphology and hearing function[J]. J Neurosci, 2015, 35(5): 1999-2014. doi: 10.1523/JNEUROSCI.3449-14.2015

[4] Borská R, Pinková B, Réblová K, et al. Inherited ichthyoses: Molecular causes of the disease in Czech patients[J]. Orphanet J Rare Dis, 2019, 14(1): 92. doi: 10.1186/s13023-019-1076-7

[5] Ma J, Ma X, Lin K, et al. Genetic screening of a Chinese cohort of children with hearing loss using a next-generation sequencing panel[J]. Hum Genomics, 2023, 17(1): 1. doi: 10.1186/s40246-022-00449-1

[6] Skau CT, Waterman CM. Specification of architecture and function of actin structures by actin nucleation factors[J]. Annu Rev Biophys, 2015, 44(1): 285-310. doi: 10.1146/annurev-biophys-060414-034308

[7] Jahan I, Elliott KL, Fritzsch B. Understanding molecular evolution and development of the organ of Corti can provide clues for hearing restoration[J]. Integr Comp Biol, 2018, 58(2): 351-365. doi: 10.1093/icb/icy019

[8] Chiereghin C, Robusto M, Massa V, et al. Role of cytoskeletal diaphanous-related formins in hearing loss[J]. Cells, 2022, 11(11): 1726. doi: 10.3390/cells11111726

[9] Morgan CP, Krey JF, Grati M, et al. PDZD7-MYO7A complex identified in enriched stereocilia membranes[J]. eLife, 2016, 5: e18312. doi: 10.7554/eLife.18312

[10] Miyoshi T, Belyantseva IA, Kitajiri SI, et al. Human deafness-associated variants alter the dynamics of key molecules in hair cell stereocilia F-actin cores[J]. Hum Genet, 2022, 141(3-4): 363-382. doi: 10.1007/s00439-021-02304-0

[11] Petersen MB, Willems PJ. Non-syndromic, autosomal-recessive deafness[J]. Clin Genet, 2006, 69(5): 371-392. doi: 10.1111/j.1399-0004.2006.00613.x

[12] Zhu M, Yang T, Wei S, et al. Mutations in the γ-actin gene (ACTG1) are associated with dominant progressive deafness (DFNA20/26)[J]. Am J Hum Genet, 2003, 73(5): 1082-1091. doi: 10.1086/379286

[13] Naz S, Griffith AJ, Riazuddin S, et al. Mutations of ESPN cause autosomal recessive deafness and vestibular dysfunction[J]. J Med Genet, 2004, 41(8): 591-595. doi: 10.1136/jmg.2004.018523

[14] Geng R, Furness DN, Muraleedharan CK, et al. The microRNA-183/96/182 cluster is essential for stereociliary bundle formation and function of cochlear sensory hair cells[J]. Sci Rep, 2018, 8(1): 18022. doi: 10.1038/s41598-018-36894-z

[15] Jiang T, Kindt K, Wu DK. Transcription factor Emx2 controls stereociliary bundle orientation of sensory hair cells[J]. eLife, 2017, 6: e23661. doi: 10.7554/eLife.23661

[16] Du X, Zadoorian A, Lukmantara IE, et al. Oxysterol-binding protein-related protein 5 (ORP5) promotes cell proliferation by activation of mTORC1 signaling[J]. J Biol Chem, 2018, 293(10): 3806-3818. doi: 10.1074/jbc.RA117.001558

[17] Ngo MH, Colbourne TR, Ridgway ND. Functional implications of sterol transport by the oxysterol-binding protein gene family[J]. Biochem J, 2010, 429(1): 13-24. doi: 10.1042/BJ20100263

[18] Koh YI, Oh KS, Kim JA, et al. OSBPL2 mutations impair autophagy and lead to hearing loss, potentially remedied by rapamycin[J]. Autophagy, 2022, 18(11): 2593-2614. doi: 10.1080/15548627.2022.2040891

[19] Xing G, Yao J, Wu B, et al. Identification of OSBPL2 as a novel candidate gene for progressive nonsyndromic hearing loss by whole-exome sequencing[J]. Genet Med, 2015, 17(3): 210-218. doi: 10.1038/gim.2014.90

[20] Thoenes M, Zimmermann U, Ebermann I, et al. OSBPL2 encodes a protein of inner and outer hair cell stereocilia and is mutated in autosomal dominant hearing loss (DFNA67)[J]. Orphanet J Rare Dis, 2015, 10(1): 15. doi: 10.1186/s13023-015-0238-5

[21] Wu N, Husile H, Yang L, et al. A novel pathogenic variant in OSBPL2 linked to hereditary late-onset deafness in a Mongolian family[J]. BMC Med Genet, 2019, 20(1): 43. doi: 10.1186/s12881-019-0781-3

[22] Yao J, Zeng H, Zhang M, et al. OSBPL2-disrupted pigs recapitulate dual features of human hearing loss and hypercholesterolaemia[J]. J Genet Genomics, 2019, 46(8): 379-387. doi: 10.1016/j.jgg.2019.06.006

[23] Shi H, Wang H, Zhang C, et al. Mutations in OSBPL2 cause hearing loss associated with primary cilia defects via sonic hedgehog signaling[J]. JCI Insight, 2022, 7(4): e149626. doi: 10.1172/jci.insight.149626

[24] Kentala H, Koponen A, Kivelä AM, et al. Analysis of ORP2-knockout hepatocytes uncovers a novel function in actin cytoskeletal regulation[J]. FASEB J, 2018, 32(3): 1281-1295. doi: 10.1096/fj.201700604R

[25] Wang H, Lin C, Yao J, et al. Deletion of OSBPL2 in auditory cells increases cholesterol biosynthesis and drives reactive oxygen species production by inhibiting AMPK activity[J]. Cell Death Dis, 2019, 10(9): 627. doi: 10.1038/s41419-019-1858-9

[26] Wang M, Dong Y, Gao S, et al. Hippo/YAP signaling pathway protects against neomycin-induced hair cell damage in the mouse cochlea[J]. Cell Mol Life Sci, 2022, 79(2): 79. doi: 10.1007/s00018-021-04029-9

[27] Liu Y, Qi J, Chen X, et al. Critical role of spectrin in hearing development and deafness[J]. Sci Adv, 2019, 5(4): eaav7803. doi: 10.1126/sciadv.aav7803

[28] Pietrangelo A, Ridgway ND. Bridging the molecular and biological functions of the oxysterol-binding protein family[J]. Cell Mol Life Sci, 2018, 75(17): 3079-3098. doi: 10.1007/s00018-018-2795-y

[29] Kentala H, Weber-Boyvat M, Olkkonen VM. OSBP-related protein family: Mediators of lipid transport and signaling at membrane contact sites[J]. Int Rev Cell Mol Biol, 2016, 321: 299-340.

[30] Dong R, Saheki Y, Swarup S, et al. Endosome-ER contacts control actin nucleation and retromer function through VAP-dependent regulation of PI4P[J]. Cell, 2016, 166(2): 408-423. doi: 10.1016/j.cell.2016.06.037

[31] Lehto M, Mäyränpää MI, Pellinen T, et al. The R-Ras interaction partner ORP3 regulates cell adhesion[J]. J Cell Sci, 2008, 121(Pt 5): 695-705.

[32] Charman M, Colbourne TR, Pietrangelo A, et al. Oxysterol-binding protein (OSBP)-related protein 4 (ORP4) is essential for cell proliferation and survival[J]. J Biol Chem, 2014, 289(22): 15705-15717. doi: 10.1074/jbc.M114.571216

[33] Guo X, Zhang L, Fan Y, et al. Oxysterol-binding protein-related protein 8 inhibits gastric cancer growth through induction of ER stress, inhibition of Wnt signaling, and activation of apoptosis[J]. Oncol Res, 2017, 25(5): 799-808. doi: 10.3727/096504016X14783691306605

[34] Perttilä J, Merikanto K, Naukkarinen J, et al. OSBPL10, a novel candidate gene for high triglyceride trait in dyslipidemic Finnish subjects, regulates cellular lipid metabolism[J]. J Mol Med (Berl), 2009, 87(8): 825-835. doi: 10.1007/s00109-009-0490-z

[35] Olkkonen VM, Ikonen E. Cholesterol transport in the late endocytic pathway: Roles of ORP family proteins[J]. J Steroid Biochem Mol Biol, 2022, 216: 106040. doi: 10.1016/j.jsbmb.2021.106040

[36] Olkkonen VM, Koponen A, Arora A. OSBP-related protein 2 (ORP2): Unraveling its functions in cellular lipid/carbohydrate metabolism, signaling and F-actin regulation[J]. J Steroid Biochem Mol Biol, 2019, 192: 105298. doi: 10.1016/j.jsbmb.2019.01.016

[37] Li D, Dammer EB, Lucki NC, et al. cAMP-stimulated phosphorylation of diaphanous 1 regulates protein stability and interaction with binding partners in adrenocortical cells[J]. Mol Biol Cell, 2013, 24(6): 848-857. doi: 10.1091/mbc.e12-08-0597

[38] Shi H, Wang H, Yao J, et al. Comparative transcriptome analysis of auditory OC-1 cells and zebrafish inner ear tissues in the absence of human OSBPL2 orthologues[J]. Biochem Biophys Res Commun, 2020, 521(1): 42-49. doi: 10.1016/j.bbrc.2019.10.061

[39] Svensmark JH, Brakebusch C. Rho GTPases in cancer: Friend or foe?[J]. Oncogene, 2019, 38(50): 7447-7456. doi: 10.1038/s41388-019-0963-7

[40] Mosaddeghzadeh N, Ahmadian MR. The RHO family GTPases: Mechanisms of regulation and signaling[J]. Cells, 2021, 10(7): 1831. doi: 10.3390/cells10071831

[41] Hall A. Rho family GTPases[J]. Biochem Soc Trans, 2012, 40(6): 1378-1382. doi: 10.1042/BST20120103

[42] Harris SL, Kazmierczak M, Pangršič T, et al. Conditional deletion of pejvakin in adult outer hair cells causes progressive hearing loss in mice[J]. Neuroscience, 2017, 344: 380-393. doi: 10.1016/j.neuroscience.2016.12.055

[43] Han Y, Wang X, Chen J, et al. Noise-induced cochlear F-actin depolymerization is mediated via ROCK2/p-ERM signaling[J]. J Neurochem, 2015, 133(5): 617-628. doi: 10.1111/jnc.13061

[44] Tsukita S, Yonemura S. ERM (ezrin/radixin/moesin) family: From cytoskeleton to signal transduction[J]. Curr Opin Cell Biol, 1997, 9(1): 70-75. doi: 10.1016/S0955-0674(97)80154-8

[45] Clucas J, Valderrama F. ERM proteins in cancer progression[J]. J Cell Sci, 2014, 127(Pt 2): 267-275. doi: 10.1242/jcs.133108

[46] Kawaguchi K, Asano S. Pathophysiological roles of actin-binding scaffold protein, ezrin[J]. Int J Mol Sci, 2022, 23(6): 3246. doi: 10.3390/ijms23063246

[47] Ng T, Parsons M, Hughes WE, et al. Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility[J]. EMBO J, 2001, 20(11): 2723-2741. doi: 10.1093/emboj/20.11.2723

[48] Zhang C, Wu Y, Xuan Z, et al. p38MAPK, Rho/ROCK and PKC pathways are involved in influenza-induced cytoskeletal rearrangement and hyperpermeability in PMVEC via phosphorylating ERM[J]. Virus Res, 2014, 192: 6-15. doi: 10.1016/j.virusres.2014.07.027

[49] Belkina NV, Liu Y, Hao J, et al. LOK is a major ERM kinase in resting lymphocytes and regulates cytoskeletal rearrangement through ERM phosphorylation[J]. Proc Natl Acad Sci U S A, 2009, 106(12): 4707-4712. doi: 10.1073/pnas.0805963106

[50] Ivetic A, Ridley AJ. Ezrin/radixin/moesin proteins and Rho GTPase signalling in leucocytes[J]. Immunology, 2004, 112(2): 165-176. doi: 10.1111/j.1365-2567.2004.01882.x

[51] Khan SY, Ahmed ZM, Shabbir MI, et al. Mutations of the RDX gene cause nonsyndromic hearing loss at the DFNB24 locus[J]. Hum Mutat, 2007, 28(5): 417-423. doi: 10.1002/humu.20469

[52] Prasad S, Vona B, Diñeiro M, et al. Radixin modulates the function of outer hair cell stereocilia[J]. Commun Biol, 2020, 3(1): 792. doi: 10.1038/s42003-020-01506-y

[53] Kitajiri S, Fukumoto K, Hata M, et al. Radixin deficiency causes deafness associated with progressive degeneration of cochlear stereocilia[J]. J Cell Biol, 2004, 166(4): 559-570. doi: 10.1083/jcb.200402007