**4. The roles of anchoring proteins in the pathogenesis of otoconia-related imbalance and dizziness/vertigo**

The inner ear acellular membranes, namely the otoconial membranes in the utricule and saccule, the cupula in the ampulla, and the tectorial membrane in the cochlea, cover their corresponding sensory epithelia, have contact with the stereocilia of hair cells and thus play crutial role in mechanotransduction. In the utricle and saccule, otoconia crystals are attached to and partially embedded in a honeycomb layer above a fibrous meshwork, which are collectively called otoconial membranes, and are responsible for the site-specific anchoring of otoconia. Disruption of the otoconial membrane structure may cause the detachment and dislocation of otoconia and thus vestibular disorders.

The acellular structures of the inner ear consist of collagenous and non-collagenous glycoproteins and proteoglycans. Several types of collagen, including type II, IV, V and IX, have been identified in the mammalian tectorial membrane (Richardson et al. 1987; Slepecky et al. 1992). In the otoconial membranes, however, otolin is likely the main collagenous component. As to the noncollagenous constituents, three glycoproteins, otogelin, α-tectorin and β-tectorin, have been identified in the inner ear acellular membranes in mice to date (Cohen-Salmon et al. 1997; Legan et al. 1997). The proteoglycan in mouse otoconia is keratin sulfate proteoglycan (KSPG) (Xu et al. 2010).

Otogelin is a glycoprotein that is present and restricted to all acellular membranes of the inner ear (Cohen-Salmon et al. 1997). At early embryonic stages, otogelin is produced by the supporting cells of the sensory epithelia of the developing vestibule and cochlea, and presents a complementary distribution pattern with Myosin VIIA, a marker of hair cells and precursors (El-Amraoui et al. 2001). At adult stages, otogelin is still expressed in the vestibular supporting cells, but become undetectable in the cochlear cells. Otogelin may be required for the attachment of the otoconial membranes and consequently site-specific anchoring of otoconia crystals. Dysfunction of otogelin in either the *Otog* knockout mice or

Proteins Involved in Otoconia Formation and Maintenance 15

of their functions. Animal models with targeted disruption of otolin and otoancorin are not yet available, and animal models with double mutant genes (e.g. Oc90 and Sc1) have not been studied but can yield more information on the precise role of the organic matrix in CaCO3 nucleation and growth. Additional studies are needed to further uncover the mechanisms underlying the spatial specific formation of otoconia. The high prevalence and debilitating nature of otoconia-related dizziness/vertigo and balance disorders necessitate these types of

The work was supported by grants from the National Institute on Deafness and Other

Anken RH, Beier M, Rahmann H. Hypergravity decreases carbonic anhydrase-reactivity in inner ear maculae of fish. J.Exp.Zoolog.A Comp Exp.Biol. 301:815-819, 2004. Anniko M, Wenngren BI, Wroblewski R. Aberrant elemental composition of otoconia in the

Banfi B, Malgrange B, Knisz J, Steger K, Dubois-Dauphin M, Krause KH. NOX3, a

Bedard K and Krause KH. The NOX family of ROS-generating NADPH oxidases:

Bianco P, Hayashi Y, Silvestrini G, Termine JD, Bonucci E. Osteonectin and Gla-protein in

Bolander ME, Young MF, Fisher LW, Yamada Y, Termine JD. Osteonectin cDNA sequence

Carlstrom D. Crystallographic study of vertebrate otoliths. Biological Bulletin 125:441-463, 1963. Cheng G, Cao Z, Xu X, van Meir EG, Lambeth JD. Homologs of gp91phox: cloning and tissue expression of Nox3, Nox4, and Nox5. Gene 269:131-140, 2001. Chun YH, Yamakoshi Y, Kim JW, Iwata T, Hu JC, Simmer JP. Porcine SPARC: isolation from

Cohen-Salmon M, El-Amraoui A, Leibovici M, Petit C. Otogelin: a glycoprotein specific to the acellular membranes of the inner ear. Proc.Natl.Acad.Sci.U.S.A 94:14450-14455, 1997. Crouch JJ and Schulte BA. Identification and cloning of site C splice variants of plasma membrane Ca-ATPase in the gerbil cochlea. Hear.Res. 101:55-61, 1996. Dai P, Stewart AK, Chebib F, Hsu A, Rozenfeld J, Huang D, Kang D, Lip V, Fang H, Shao H,

physiology and pathophysiology. Physiol Rev. 87:245-313, 2007.

dancer mouse mutant with a semidominant gene causing a morphogenetic type of

superoxide-generating NADPH oxidase of the inner ear. J.Biol.Chem. 279:46065-

calf bone: ultrastructural immunohistochemical localization using the Protein A-

reveals potential binding regions for calcium and hydroxyapatite and shows homologies with both a basement membrane protein (SPARC) and a serine proteinase inhibitor (ovomucoid). Proc.Natl.Acad.Sci.U.S.A 85:2919-2923, 1988. Boskey AL, Maresca M, Ullrich W, Doty SB, Butler WT, Prince CW. Osteopontin-

hydroxyapatite interactions in vitro: inhibition of hydroxyapatite formation and

dentin, cDNA sequence, and computer model. Eur.J.Oral Sci. 114 Suppl 1:78-85, 2006.

Liu X, Yu F, Yuan H, Kenna M, Miller DT, Shen Y, Yang W, Zelikovic I, Platt OS, Han D, Alper SL, Wu BL. Distinct and novel SLC26A4/Pendrin mutations in Chinese and U.S. patients with nonsyndromic hearing loss. Physiol Genomics

studies as they are the foundation required to uncover the molecular etiology.

Communication Disorders (R01 DC008603 and DC008603-S1 to Y.W.L.).

inner ear defect. Acta Otolaryngol. 106:208-212, 1988.

gold method. Calcif.Tissue Int. 37:684-686, 1985.

growth in a gelatin-gel. Bone Miner. 22:147-159, 1993.

**6. Acknowledgements** 

46072, 2004.

38:281-290, 2009.

**7. References** 

the twister mutant mice leads to severe vestibular deficits, which is postulated to be caused by displaced otoconial membranes in the utricle and saccule (Simmler et al. 2000a; Simmler et al. 2000b).

α-tectorin and β-tectorin, named with reference to their localization, are major noncollagenous glycoproteins of the mammalian tectorial membrane (Legan et al. 1997). In addition, these two proteins are abundant constituents of the otoconial membranes, but are not present in the cupula (Goodyear and Richardson 2002; Xu et al. 2010). In the mouse vestibule, α-tectorin is mainly expressed between E12.5 and P15 in the transitional zone, as well as in a region that is producing the accessory membranes of the utricle and saccule, but absent in the ampullae of semicircular canals (Rau et al. 1999). Mice with targeted deletion of α-tectorin display reduced otoconial membranes and a few scattered giant otoconia (Legan et al. 2000).

β-tectorin has a spatial and temporal expression pattern distinct from that of α-tectorin in the vestibule. It is expressed in the striolar region of the utricule and saccule from E14.5 until at least P150 (Legan et al. 1997; Rau et al. 1999), suggesting that the striolar and extrastriolar region of the otoconial membranes may have different composition. *Tectb* null mice show structural disruption of the tectorial membrane and hearing loss at low frequencies (Russell et al. 2007). However, no vestibular defects have been reported.

Interestingly, both otogelin and α-tectorin possess several von Willebrand factor type D (VWFD) domains containing the multimerization consensus site CGLC (Mayadas and Wagner 1992). This structural feature is probably essential for the multimer assembly of those proteins to form filament and higher order structures.

Otoancorin is a glycosylphosphatidylinositol (GPI)-anchored protein specific to the interface between the sensory epithelia and their overlying acellular membranes of the inner ear (Zwaenepoel et al. 2002). In the vestibule, otoancorin is expressed on the apical surface of the supporting cells in the utricle, saccule and crista. Although the function of otoancorin has not been elucidated, the C-terminal GPI anchor motif of this protein likely facilitates the otoancorin-cell surface adhesion. It is proposed that otoancorin may interact with the other components of the otoconial membranes, such as otogelin and tectorins, and with the epithelial surface, thus mediating the attachment of otoconial membranes to the underlying sensory epithelia (Zwaenepoel et al. 2002).

#### **5. Summary and future direction**

Like other biominerals such as bone and teeth, otoconia primarily differ from their nonbiological counterparts by their protein-mediated nucleation, growth and maintenance processes. With only CaCO3 crystallites and less than a dozen glycoprotein/proteoglycan components, otoconia are seemingly simple biological structures compared to other tissues. Yet, the processes governing otoconia formation are multiple and involve many more molecules and much complicated cellular and extracellular events including matrix assembly, endolymph homeostasis and proper function of ion channels/pumps. Expression of the involved genes is well orchestrated temporally and spatially, and the functions of their proteins are finely coordinated for optimal crystal formation. Some of these proteins also play vital roles in normal cellular activities (e.g. hair cell stimulation) and other vestibular function. Some other proteins (e.g. otolin, tectorins and otoancorin) still need to be further investigated of their functions. Animal models with targeted disruption of otolin and otoancorin are not yet available, and animal models with double mutant genes (e.g. Oc90 and Sc1) have not been studied but can yield more information on the precise role of the organic matrix in CaCO3 nucleation and growth. Additional studies are needed to further uncover the mechanisms underlying the spatial specific formation of otoconia. The high prevalence and debilitating nature of otoconia-related dizziness/vertigo and balance disorders necessitate these types of studies as they are the foundation required to uncover the molecular etiology.

#### **6. Acknowledgements**

The work was supported by grants from the National Institute on Deafness and Other Communication Disorders (R01 DC008603 and DC008603-S1 to Y.W.L.).

#### **7. References**

14 Otolaryngology

the twister mutant mice leads to severe vestibular deficits, which is postulated to be caused by displaced otoconial membranes in the utricle and saccule (Simmler et al. 2000a; Simmler

α-tectorin and β-tectorin, named with reference to their localization, are major noncollagenous glycoproteins of the mammalian tectorial membrane (Legan et al. 1997). In addition, these two proteins are abundant constituents of the otoconial membranes, but are not present in the cupula (Goodyear and Richardson 2002; Xu et al. 2010). In the mouse vestibule, α-tectorin is mainly expressed between E12.5 and P15 in the transitional zone, as well as in a region that is producing the accessory membranes of the utricle and saccule, but absent in the ampullae of semicircular canals (Rau et al. 1999). Mice with targeted deletion of α-tectorin display reduced otoconial membranes and a few scattered giant otoconia

β-tectorin has a spatial and temporal expression pattern distinct from that of α-tectorin in the vestibule. It is expressed in the striolar region of the utricule and saccule from E14.5 until at least P150 (Legan et al. 1997; Rau et al. 1999), suggesting that the striolar and extrastriolar region of the otoconial membranes may have different composition. *Tectb* null mice show structural disruption of the tectorial membrane and hearing loss at low frequencies (Russell

Interestingly, both otogelin and α-tectorin possess several von Willebrand factor type D (VWFD) domains containing the multimerization consensus site CGLC (Mayadas and Wagner 1992). This structural feature is probably essential for the multimer assembly of

Otoancorin is a glycosylphosphatidylinositol (GPI)-anchored protein specific to the interface between the sensory epithelia and their overlying acellular membranes of the inner ear (Zwaenepoel et al. 2002). In the vestibule, otoancorin is expressed on the apical surface of the supporting cells in the utricle, saccule and crista. Although the function of otoancorin has not been elucidated, the C-terminal GPI anchor motif of this protein likely facilitates the otoancorin-cell surface adhesion. It is proposed that otoancorin may interact with the other components of the otoconial membranes, such as otogelin and tectorins, and with the epithelial surface, thus mediating the attachment of otoconial membranes to the underlying

Like other biominerals such as bone and teeth, otoconia primarily differ from their nonbiological counterparts by their protein-mediated nucleation, growth and maintenance processes. With only CaCO3 crystallites and less than a dozen glycoprotein/proteoglycan components, otoconia are seemingly simple biological structures compared to other tissues. Yet, the processes governing otoconia formation are multiple and involve many more molecules and much complicated cellular and extracellular events including matrix assembly, endolymph homeostasis and proper function of ion channels/pumps. Expression of the involved genes is well orchestrated temporally and spatially, and the functions of their proteins are finely coordinated for optimal crystal formation. Some of these proteins also play vital roles in normal cellular activities (e.g. hair cell stimulation) and other vestibular function. Some other proteins (e.g. otolin, tectorins and otoancorin) still need to be further investigated

et al. 2007). However, no vestibular defects have been reported.

those proteins to form filament and higher order structures.

sensory epithelia (Zwaenepoel et al. 2002).

**5. Summary and future direction** 

et al. 2000b).

(Legan et al. 2000).


Proteins Involved in Otoconia Formation and Maintenance 17

Hohenester E, Maurer P, Timpl R. Crystal structure of a pair of follistatin-like and EF-hand

Hohenester E, Sasaki T, Giudici C, Farndale RW, Bachinger HP. Structural basis of

Hughes I, Blasiole B, Huss D, Warchol ME, Rath NP, Hurle B, Ignatova E, Dickman JD,

Hughes I, Saito M, Schlesinger PH, Ornitz DM. Otopetrin 1 activation by purinergic nucleotides regulates intracellular calcium. Proc.Natl.Acad.Sci.U.S.A 104:12023-12028, 2007. Hunter GK, Kyle CL, Goldberg HA. Modulation of crystal formation by bone

Hurle B, Ignatova E, Massironi SM, Mashimo T, Rios X, Thalmann I, Thalmann R, Ornitz DM.

Iozzo RV. Matrix proteoglycans: from molecular design to cellular function.

Ishibashi T, Takumida M, Akagi N, Hirakawa K, Anniko M. Expression of transient receptor

Ito M, Spicer SS, Schulte BA. Histochemical detection of glycogen and glycoconjugates in the

Jahnen-Dechent W, Schinke T, Trindl A, Muller-Esterl W, Sablitzky F, Kaiser S, Blessing M.

Johnston IG, Paladino T, Gurd JW, Brown IR. Molecular cloning of SC1: a putative brain

Jones SM, Erway LC, Bergstrom RA, Schimenti JC, Jones TA. Vestibular responses to linear

Jones SM, Erway LC, Johnson KR, Yu H, Jones TA. Gravity receptor function in mice with

Kang YJ, Stevenson AK, Yau PM, Kollmar R. Sparc protein is required for normal growth of

Kaufmann B, Muller S, Hanisch FG, Hartmann U, Paulsson M, Maurer P, Zaucke F.

Keeton TP, Burk SE, Shull GE. Alternative splicing of exons encoding the calmodulin-

Kido T, Sekitani T, Yamashita H, Endo S, Masumitsu Y, Shimogori H. Effects of carbonic

Kim E, Hyrc KL, Speck J, Lundberg YW, Salles FT, Kachar B, Goldberg MP, Warchol ME,

graded otoconial deficiencies. Hear.Res. 191:34-40, 2004.

collagen affinity. Glycobiology 14:609-619, 2004.

3, and 4. J.Biol.Chem. 268:2740-2748, 1993.

Am.J.Otolaryngol. 12:191-195, 1991.

Otopetrin 1. J.Neurophysiol.2010.

zebrafish otoliths. J.Assoc.Res.Otolaryngol. 9:436-451, 2008.

sequence-specific collagen recognition by SPARC. Proc.Natl.Acad.Sci.U.S.A

Thalmann R, Levenson R, Ornitz DM. Otopetrin 1 is required for otolith formation

phosphoproteins: structural specificity of the osteopontin-mediated inhibition of

Non-syndromic vestibular disorder with otoconial agenesis in tilted/mergulhador mice

potential vanilloid (TRPV) 1, 2, 3, and 4 in mouse inner ear. Acta Otolaryngol.

inner ear with modified concanavalin A-horseradish peroxidase procedures.

Cloning and targeted deletion of the mouse fetuin gene. J.Biol.Chem. 272:31496-

extracellular matrix glycoprotein showing partial similarity to osteonectin/BM40/

acceleration are absent in otoconia-deficient C57BL/6JEi-het mice. Hear.Res.

Structural variability of BM-40/SPARC/osteonectin glycosylation: implications for

binding domains and C termini of plasma membrane Ca(2+)-ATPase isoforms 1, 2,

anhydrase inhibitor on the otolithic organs of developing chick embryos.

Ornitz DM. Regulation of cellular calcium in vestibular supporting cells by

calcium-binding domains in BM-40. EMBO J. 16:3778-3786, 1997.

in the zebrafish Danio rerio. Dev.Biol. 276:391-402, 2004.

hydroxyapatite formation. Biochem.J. 300 ( Pt 3):723-728, 1994.

caused by mutations in otopetrin 1. Hum.Mol.Genet. 12:777-789, 2003.

105:18273-18277, 2008.

128:1286-1293, 2008.

31503, 1997.

135:56-60, 1999.

Histochem.J. 26:437-446, 1994.

SPARC. Neuron 4:165-176, 1990.

Annu.Rev.Biochem. 67:609-652, 1998.


Deans MR, Peterson JM, Wong GW. Mammalian Otolin: a multimeric glycoprotein specific

Dou H, Xu J, Wang Z, Smith AN, Soleimani M, Karet FE, Greinwald JH, Jr., Choo D. Co-

Dror AA, Politi Y, Shahin H, Lenz DR, Dossena S, Nofziger C, Fuchs H, Hrabe de AM,

ear as a result of an Slc26a4 mutation. J.Biol.Chem. 285:21724-21735, 2010. Dumont RA, Lins U, Filoteo AG, Penniston JT, Kachar B, Gillespie PG. Plasma membrane

Duvall CL, Taylor WR, Weiss D, Wojtowicz AM, Guldberg RE. Impaired angiogenesis, early

El-Amraoui A, Cohen-Salmon M, Petit C, Simmler MC. Spatiotemporal expression of otogelin in the developing and adult mouse inner ear. Hear.Res. 158:151-159, 2001. Endo S, Sekitani T, Yamashita H, Kido T, Masumitsu Y, Ogata M, Miura M. Glycoconjugates

Everett LA, Belyantseva IA, Noben-Trauth K, Cantos R, Chen A, Thakkar SI, Hoogstraten-

Everett LA, Morsli H, Wu DK, Green ED. Expression pattern of the mouse ortholog of the

Fermin CD, Lovett AE, Igarashi M, Dunner K, Jr. Immunohistochemistry and histochemistry

Ferrary E, Tran Ba HP, Roinel N, Bernard C, Amiel C. Calcium and the inner ear fluids. Acta

Furuta H, Luo L, Hepler K, Ryan AF. Evidence for differential regulation of calcium by

George A, Sabsay B, Simonian PA, Veis A. Characterization of a novel dentin matrix acidic

Goodyear RJ and Richardson GP. Extracellular matrices associated with the apical surfaces

Hassell J, Yamada Y, rikawa-Hirasawa E. Role of perlecan in skeletal development and

Heiss A, DuChesne A, Denecke B, Grotzinger J, Yamamoto K, Renne T, Jahnen-Dechent W.

Cerebellin-1. PLoS.ONE. 5:e12765-2010.

J.Histochem.Cytochem. 52:1377-1384, 2004.

deficient mice. J.Bone Miner.Res. 22:286-297, 2007.

481:116-120, 1991.

Hum.Mol.Genet. 10:153-161, 2001.

Otolaryngol.Suppl 460:13-17, 1988.

Hear.Res. 123:10-26, 1998.

J.Neurobiol. 53:212-227, 2002.

diseases. Glycoconj.J. 19:263-267, 2002.

268:12624-12630, 1993.

Proc.Natl.Acad.Sci.U.S.A 96:9727-9732, 1999.

domesticus). Acta Anat.(Basel) 138:75-83, 1990.

to the inner ear that interacts with otoconial matrix protein Otoconin-90 and

expression of pendrin, vacuolar H+-ATPase alpha4-subunit and carbonic anhydrase II in epithelial cells of the murine endolymphatic sac.

Paulmichl M, Weiner S, Avraham KB. Calcium oxalate stone formation in the inner

Ca2+-ATPase isoform 2a is the PMCA of hair bundles. J.Neurosci. 21:5066-5078, 2001.

callus formation, and late stage remodeling in fracture healing of osteopontin-

in the otolithic organ of the developing chick embryo. Acta Otolaryngol.Suppl

Miller SL, Kachar B, Wu DK, Green ED. Targeted disruption of mouse Pds provides insight about the inner-ear defects encountered in Pendred syndrome.

Pendred's syndrome gene (Pds) suggests a key role for pendrin in the inner ear.

of the inner ear gelatinous membranes and statoconia of the chick (Gallus

outer versus inner hair cells: plasma membrane Ca-ATPase gene expression.

phosphoprotein. Implications for induction of biomineralization. J.Biol.Chem.

of sensory epithelia in the inner ear: molecular and structural diversity.

Structural basis of calcification inhibition by alpha 2-HS glycoprotein/fetuin-A. Formation of colloidal calciprotein particles. J.Biol.Chem. 278:13333-13341, 2003. Hirst KL, Ibaraki-O'Connor K, Young MF, Dixon MJ. Cloning and expression analysis of the bovine dentin matrix acidic phosphoprotein gene. J.Dent.Res. 76:754-760, 1997.


Proteins Involved in Otoconia Formation and Maintenance 19

Mann S, Parker SB, Ross MD, Skarnulis AJ, Williams RJ. The ultrastructure of the calcium

Marcus DC and Wangemann P. Cochlear and Vestibular Function and Dysfunction. In

Maurer P, Hohenadl C, Hohenester E, Gohring W, Timpl R, Engel J. The C-terminal portion

domain that binds calcium and collagen IV. J.Mol.Biol. 253:347-357, 1995. Mayadas TN and Wagner DD. Vicinal cysteines in the prosequence play a role in von

McKinnon PJ and Margolskee RF. SC1: a marker for astrocytes in the adult rodent brain is upregulated during reactive astrocytosis. Brain Res. 709:27-36, 1996. McKinnon PJ, McLaughlin SK, Kapsetaki M, Margolskee RF. Extracellular matrix-associated

Mendis DB and Brown IR. Expression of the gene encoding the extracellular matrix

Murayama E, Herbomel P, Kawakami A, Takeda H, Nagasawa H. Otolith matrix proteins

Murayama E, Takagi Y, Nagasawa H. Immunohistochemical localization of two otolith

Nakano Y, Banfi B, Jesaitis AJ, Dinauer MC, Allen LA, Nauseef WM. Critical roles for

Nakano Y, Longo-Guess CM, Bergstrom DE, Nauseef WM, Jones SM, Banfi B. Mutation of

Nakaya K, Harbidge DG, Wangemann P, Schultz BD, Green ED, Wall SM, Marcus DC. Lack

Oldberg A, Franzen A, Heinegard D. Cloning and sequence analysis of rat bone sialoprotein

Paffenholz R, Bergstrom RA, Pasutto F, Wabnitz P, Munroe RJ, Jagla W, Heinzmann U,

Pedrozo HA, Schwartz Z, Dean DD, Harrison JL, Campbell JW, Wiederhold ML, Boyan BD.

anchoring onto the sensory maculae. Mech.Dev. 122:791-803, 2005.

microscopy study. Proc.R.Soc.Lond B Biol.Sci. 218:415-424, 1983.

2009:421-433.

Res. 24:11-19, 1994.

403:97-108, 2007.

Histochem.Cell Biol. 121:155-166, 2004.

J.Clin.Invest 118:1176-1185, 2008.

Physiol 292:F1314-F1321, 2007.

Calcif.Tissue Int. 61:247-255, 1997.

Proc.Natl.Acad.Sci.U.S.A 83:8819-8823, 1986.

NADPH oxidase. Genes Dev. 18:486-491, 2004.

carbonate balance organs of the inner ear: an ultra-high resolution electron

*Physiology and Pathology of Chloride Transporters and Channels in the Nervous System-- From molecules to diseases.* Edited by Alvarez-Leefmans FJ, Delpire E. Elsevier;

of BM-40 (SPARC/osteonectin) is an autonomously folding and crystallisable

Willebrand factor multimer assembly. Proc.Natl.Acad.Sci.U.S.A 89:3531-3535, 1992.

protein Sc1 is not essential for mouse development. Mol.Cell Biol. 20:656-660, 2000.

glycoprotein SPARC in the developing and adult mouse brain. Brain Res.Mol.Brain

OMP-1 and Otolin-1 are necessary for normal otolith growth and their correct

matrix proteins in the otolith and inner ear of the rainbow trout, Oncorhynchus mykiss: comparative aspects between the adult inner ear and embryonic otocysts.

p22phox in the structural maturation and subcellular targeting of Nox3. Biochem.J.

the Cyba gene encoding p22phox causes vestibular and immune defects in mice.

of pendrin HCO3- transport elevates vestibular endolymphatic [Ca2+] by inhibition of acid-sensitive TRPV5 and TRPV6 channels. Am.J.Physiol Renal

(osteopontin) cDNA reveals an Arg-Gly-Asp cell-binding sequence.

Marquardt A, Bareiss A, Laufs J, Russ A, Stumm G, Schimenti JC, Bergstrom DE. Vestibular defects in head-tilt mice result from mutations in Nox3, encoding an

Evidence for the involvement of carbonic anhydrase and urease in calcium carbonate formation in the gravity-sensing organ of Aplysia californica.


Kim E, Hyrc KL, Speck J, Salles FT, Lundberg YW, Goldberg MP, Kachar B, Warchol ME,

Kim HM and Wangemann P. Failure of fluid absorption in the endolymphatic sac initiates

Kim HM and Wangemann P. Epithelial cell stretching and luminal acidification lead to a

Kishore U and Reid KB. Modular organization of proteins containing C1q-like globular

Kiss PJ, Knisz J, Zhang Y, Baltrusaitis J, Sigmund CD, Thalmann R, Smith RJ, Verpy E, Banfi

Kozel PJ, Friedman RA, Erway LC, Yamoah EN, Liu LH, Riddle T, Duffy JJ, Doetschman T,

Kumagami H, Terakado M, Sainoo Y, Baba A, Fujiyama D, Fukuda T, Takasaki K, Takahashi

Legan PK, Lukashkina VA, Goodyear RJ, Kossi M, Russell IJ, Richardson GP. A targeted

Legan PK, Rau A, Keen JN, Richardson GP. The mouse tectorins. Modular matrix proteins of

Lim DJ. Otoconia in health and disease. A review. Ann.Otol.Rhinol.Laryngol.Suppl 112:17-

Lim DJ, Karabinas C, Trune DR. Histochemical localization of carbonic anhydrase in the

Lins U, Farina M, Kurc M, Riordan G, Thalmann R, Thalmann I, Kachar B. The otoconia of

Lively S, Ringuette MJ, Brown IR. Localization of the extracellular matrix protein SC1 to

Lu W, Zhou D, Freeman JJ, Thalmann I, Ornitz DM, Thalmann R. In vitro effects of

Luxon LM, Cohen M, Coffey RA, Phelps PD, Britton KE, Jan H, Trembath RC, Reardon W. Neuro-otological findings in Pendred syndrome. Int.J.Audiol. 42:82-88, 2003. Lv K, Huang H, Lu Y, Qin C, Li Z, Feng JQ. Circling behavior developed in Dmp1 null mice is due to bone defects in the vestibular apparatus. Int.J.Biol.Sci. 6:537-545, 2010. MacDougall M, Gu TT, Luan X, Simmons D, Chen J. Identification of a novel isoform of

synapses in the adult rat brain. Neurochem.Res. 32:65-71, 2007.

46:655-661, 2011.

PLoS.ONE. 5:e14041-2010.

PLoS.ONE. 6:e17949-2011.

Curr.Biol. 16:208-213, 2006.

J.Biol.Chem. 273:18693-18696, 1998.

J.Biol.Chem. 272:8791-8801, 1997.

inner ear. Am.J.Otolaryngol. 4:33-42, 1983.

structure. Hear.Res. 268:172-183, 2010.

Miner.Res. 13:422-431, 1998.

the filament matrix. J.Struct.Biol. 131:67-78, 2000.

24, 1984.

domain. Immunopharmacology 42:15-21, 1999.

endolymphatic sac. Audiol.Neurootol. 14:190-197, 2009.

gain and timing of cochlear feedback. Neuron 28:273-285, 2000.

Ornitz DM. Missense mutations in Otopetrin 1 affect subcellular localization and inhibition of purinergic signaling in vestibular supporting cells. Mol.Cell Neurosci.

cochlear enlargement that leads to deafness in mice lacking pendrin expression.

retarded development of stria vascularis and deafness in mice lacking pendrin.

B. Inactivation of NADPH oxidase organizer 1 Results in Severe Imbalance.

Miller ML, Cardell EL, Shull GE. Balance and hearing deficits in mice with a null mutation in the gene encoding plasma membrane Ca2+-ATPase isoform 2.

H. Expression of the osmotically responsive cationic channel TRPV4 in the

deletion in alpha-tectorin reveals that the tectorial membrane is required for the

the inner ear homologous to components of the sperm-egg adhesion system.

the guinea pig utricle: internal structure, surface exposure, and interactions with

recombinant otoconin 90 upon calcite crystal growth. Significance of tertiary

mouse dentin matrix protein 1: spatial expression in mineralized tissues. J.Bone


Proteins Involved in Otoconia Formation and Maintenance 21

Shapses SA, Cifuentes M, Spevak L, Chowdhury H, Brittingham J, Boskey AL, Denhardt

Simmler MC, Zwaenepoel I, Verpy E, Guillaud L, Elbaz C, Petit C, Panthier JJ. Twister

Slepecky NB, Savage JE, Yoo TJ. Localization of type II, IX and V collagen in the inner ear.

Sollner C, Burghammer M, Busch-Nentwich E, Berger J, Schwarz H, Riekel C, Nicolson T.

Sollner C, Schwarz H, Geisler R, Nicolson T. Mutated otopetrin 1 affects the genesis of

Squires TM, Weidman MS, Hain TC, Stone HA. A mathematical model for top-shelf vertigo: the role of sedimenting otoconia in BPPV. J.Biomech. 37:1137-1146, 2004. Steyger PS and Wiederhold ML. Visualization of newt aragonitic otoconial matrices using

Swartz DJ and Santi PA. Immunohistochemical localization of keratan sulfate in the

Takemura T, Sakagami M, Nakase T, Kubo T, Kitamura Y, Nomura S. Localization of osteopontin in the otoconial organs of adult rats. Hear.Res. 79:99-104, 1994. Takumida M, Ishibashi T, Hamamoto T, Hirakawa K, Anniko M. Age-dependent changes in

Termine JD, Kleinman HK, Whitson SW, Conn KM, McGarvey ML, Martin GR. Osteonectin, a bone-specific protein linking mineral to collagen. Cell 26:99-105, 1981. Thalmann I, Hughes I, Tong BD, Ornitz DM, Thalmann R. Microscale analysis of proteins in

Thalmann R, Ignatova E, Kachar B, Ornitz DM, Thalmann I. Development and maintenance of otoconia: biochemical considerations. Ann.N.Y.Acad.Sci. 942:162-178, 2001. Thurner PJ, Chen CG, Ionova-Martin S, Sun L, Harman A, Porter A, Ager JW, III, Ritchie

Tohse H and Mugiya Y. Effects of enzyme and anion transport inhibitors on in vitro

Trune DR and Lim DJ. The behavior and vestibular nuclear morphology of otoconia-

Tsujikawa S, Yamashita T, Tomoda K, Iwai H, Kumazawa H, Cho H, Kumazawa T. Effects

deficient pallid mutant mice. J.Neurogenet. 1:53-69, 1983.

the expression of klotho protein, TRPV5 and TRPV6 in mouse inner ear. Acta

inner ear tissues and fluids with emphasis on endolymphatic sac, otoconia, and

RO, Alliston T. Osteopontin deficiency increases bone fragility but preserves bone

incorporation of inorganic carbon and calcium into endolymph and otoliths in salmon Oncorhynchus masou. Comp Biochem.Physiol A Mol.Integr.Physiol

of acetazolamide on acid-base balance in the endolymphatic sac of the guinea pig.

transmission electron microscopy. Hear.Res. 92:184-191, 1995.

membranes. Mamm.Genome 11:960-966, 2000b.

biomineralization. Science 302:282-286, 2003.

chinchilla inner ear. Hear.Res. 109:92-101, 1997.

organ of Corti. Electrophoresis 27:1598-1608, 2006.

Otolaryngol. 129:1340-1350, 2009.

mass. Bone 46:1564-1573, 2010.

Acta Otolaryngol.Suppl 500:50-53, 1993.

128:177-184, 2001.

Acta Otolaryngol. 112:611-617, 1992.

and content during calcium deficiency. Calcif.Tissue Int. 73:86-92, 2003. Simmler MC, Cohen-Salmon M, El-Amraoui A, Guillaud L, Benichou JC, Petit C, Panthier JJ.

24:139-143, 2000a.

590, 2004.

DT. Osteopontin facilitates bone resorption, decreasing bone mineral crystallinity

Targeted disruption of otog results in deafness and severe imbalance. Nat.Genet.

mutant mice are defective for otogelin, a component specific to inner ear acellular

Control of crystal size and lattice formation by starmaker in otolith

otoliths and the localization of Starmaker in zebrafish. Dev.Genes Evol. 214:582-


Petko JA, Millimaki BB, Canfield VA, Riley BB, Levenson R. Otoc1: a novel otoconin-90

Pisam M, Jammet C, Laurent D. First steps of otolith formation of the zebrafish: role of

Pote KG and Ross MD. Each otoconia polymorph has a protein unique to that polymorph.

Price PA, Thomas GR, Pardini AW, Figueira WF, Caputo JM, Williamson MK. Discovery of

Quelch KJ, Cole WG, Melick RA. Noncollagenous proteins in normal and pathological

Rau A, Legan PK, Richardson GP. Tectorin mRNA expression is spatially and temporally restricted during mouse inner ear development. J.Comp Neurol. 405:271-280, 1999. Richardson GP, Russell IJ, Duance VC, Bailey AJ. Polypeptide composition of the

Rodan SB and Rodan GA. Integrin function in osteoclasts. J.Endocrinol. 154 Suppl:S47-S56,

Ross MD and Pote KG. Some properties of otoconia. Philos.Trans.R.Soc.Lond B Biol.Sci.

Russell IJ, Legan PK, Lukashkina VA, Lukashkin AN, Goodyear RJ, Richardson GP.

Sage EH and Vernon RB. Regulation of angiogenesis by extracellular matrix: the growth and

Sakagami M. Role of osteopontin in the rodent inner ear as revealed by in situ hybridization.

Salamat MS, Ross MD, Peacor DR. Otoconial formation in the fetal rat.

Salt AN, Inamura N, Thalmann R, Vora A. Calcium gradients in inner ear endolymph.

Salvinelli F, Firrisi L, Casale M, Trivelli M, D'Ascanio L, Lamanna F, Greco F, Costantino S.

Sasaki T, Hohenester E, Gohring W, Timpl R. Crystal structure and mapping by site-

Schafer C, Heiss A, Schwarz A, Westenfeld R, Ketteler M, Floege J, Muller-Esterl W, Schinke

Schuknecht HF. Positional vertigo: clinical and experimental observations.

Scott DA and Karniski LP. Human pendrin expressed in Xenopus laevis oocytes mediates chloride/formate exchange. Am.J.Physiol Cell Physiol 278:C207-C211, 2000. Scott DA, Wang R, Kreman TM, Sheffield VC, Karniski LP. The Pendred syndrome gene encodes a chloride-iodide transport protein. Nat.Genet. 21:440-443, 1999.

Benign paroxysmal positional vertigo: diagnosis and treatment. Clin.Ter. 155:395-

directed mutagenesis of the collagen-binding epitope of an activated form of BM-

T, Jahnen-Dechent W. The serum protein alpha 2-Heremans-Schmid glycoprotein/fetuin-A is a systemically acting inhibitor of ectopic calcification.

Sharpened cochlear tuning in a mouse with a genetically modified tectorial

glycogen? Cell Tissue Res. 310:163-168, 2002.

Comp Biochem.Physiol B 98:287-295, 1991.

human bone. Calcif.Tissue Int. 36:545-549, 1984.

membrane. Nat.Neurosci. 10:215-223, 2007.

Med.Electron Microsc. 33:3-10, 2000.

Am.J.Otolaryngol. 10:371-375, 1989.

J.Clin.Invest 112:357-366, 2003.

the glue. J.Hypertens.Suppl 12:S145-S152, 1994.

Ann.Otol.Rhinol.Laryngol. 89:229-238, 1980.

40/SPARC/osteonectin. EMBO J. 17:1625-1634, 1998.

Schuknecht HF. Cupulolithiasis. Arch.Otolaryngol. 90:765-778, 1969.

Trans.Am.Acad.Ophthalmol.Otolaryngol. 66:319-332, 1962.

mammalian tectorial membrane. Hear.Res. 25:45-60, 1987.

J.Biol.Chem. 277:3926-3934, 2002.

222, 2008.

1997.

400, 2004.

304:445-452, 1984.

ortholog required for otolith mineralization in zebrafish. Dev.Neurobiol. 68:209-

a high molecular weight complex of calcium, phosphate, fetuin, and matrix gamma-carboxyglutamic acid protein in the serum of etidronate-treated rats.


**Section 2** 

**Rhinology** 


**Section 2** 

22 Otolaryngology

Verpy E, Leibovici M, Petit C. Characterization of otoconin-95, the major protein of murine

Viviano BL, Silverstein L, Pflederer C, Paine-Saunders S, Mills K, Saunders S. Altered

secretory phospholipase A2. Proc.Natl.Acad.Sci.U.S.A 95:15345-15350, 1998. Westenfeld R, Schafer C, Kruger T, Haarmann C, Schurgers LJ, Reutelingsperger C, Ivanovski

phosphate challenge in mice. Nephrol.Dial.Transplant. 22:1537-1546, 2007. Xu T, Bianco P, Fisher LW, Longenecker G, Smith E, Goldstein S, Bonadio J, Boskey A,

Xu Y, Zhang H, Yang H, Zhao X., Lovas S, Lundberg YW. Expression, functional and

Xu Y and Lundberg Y.W. Temporally and spatially regulated expression of otoconial genes. 35th Association for Research in Otolaryngology MidWinter Meeting #282,2012. Xu Y, Yang L, Jones S.M., Zhao X., Zhang Y, Lundberg Y.W. Functional cooperation of two

Yamauchi D, Nakaya K, Raveendran NN, Harbidge DG, Singh R, Wangemann P, Marcus

Yamoah EN, Lumpkin EA, Dumont RA, Smith PJ, Hudspeth AJ, Gillespie PG. Plasma

Yang H, Zhao X, Xu Y, Wang L, He Q, Lundberg YW. Matrix recruitment and calcium sequestration for spatial specific otoconia development. PLoS.ONE. 6:e20498-2011. Young MF, Bi Y, Ameye L, Chen XD. Biglycan knockout mice: new models for

Zhao X, Jones SM, Thoreson WB, Lundberg YW. Osteopontin is not critical for otoconia formation or balance function. J.Assoc.Res.Otolaryngol. 9:191-201, 2008a. Zhao X, Jones SM, Yamoah EN, Lundberg YW. Otoconin-90 deletion leads to imbalance but

Zhao X, Yang H, Yamoah EN, Lundberg YW. Gene targeting reveals the role of Oc90 as the essential organizer of the otoconial organic matrix. Dev.Biol. 304:508-524, 2007. Zwaenepoel I, Mustapha M, Leibovici M, Verpy E, Goodyear R, Liu XZ, Nouaille S, Nance

recessive deafness DFNB22. Proc.Natl.Acad.Sci.U.S.A 99:6240-6245, 2002.

musculoskeletal diseases. Glycoconj.J. 19:257-262, 2002.

Proc.Natl.Acad.Sci.U.S.A 96:529-534, 1999.

like phenotype in mice. Nat.Genet. 20:78-82, 1998.

239:2659-2673, 2010.

18:610-624, 1998.

299, 2008b.

MidWinter Meeting #281:2012.

labyrinth. BMC.Physiol 10:1-2010.

otoconia, provides insights into the formation of these inner ear biominerals.

hematopoiesis in glypican-3-deficient mice results in decreased osteoclast differentiation and a delay in endochondral ossification. Dev.Biol. 282:152-162, 2005. Wang Y, Kowalski PE, Thalmann I, Ornitz DM, Mager DL, Thalmann R. Otoconin-90, the

mammalian otoconial matrix protein, contains two domains of homology to

O, Drueke T, Massy ZA, Ketteler M, Floege J, Jahnen-Dechent W. Fetuin-A protects against atherosclerotic calcification in CKD. J.Am.Soc.Nephrol. 20:1264-1274, 2009. Westenfeld R, Schafer C, Smeets R, Brandenburg VM, Floege J, Ketteler M, Jahnen-Dechent

W. Fetuin-A (AHSG) prevents extraosseous calcification induced by uraemia and

Heegaard AM, Sommer B, Satomura K, Dominguez P, Zhao C, Kulkarni AB, Robey PG, Young MF. Targeted disruption of the biglycan gene leads to an osteoporosis-

structural analysis of proteins critical for otoconia development. Dev.Dyn.

otoconial proteins Oc90 and Nox3. 35th Association for Research in Otolaryngology

DC. Expression of epithelial calcium transport system in rat cochlea and vestibular

membrane Ca2+-ATPase extrudes Ca2+ from hair cell stereocilia. J.Neurosci.

normal hearing: a comparison with other otoconia mutants. Neuroscience 153:289-

WE, Kanaan M, Avraham KB, Tekaia F, Loiselet J, Lathrop M, Richardson G, Petit C. Otoancorin, an inner ear protein restricted to the interface between the apical surface of sensory epithelia and their overlying acellular gels, is defective in autosomal **Rhinology** 

**2** 

**Epistaxis** 

*South Korea* 

Jin Hee Cho and Young Ha Kim

*College of Medicine, The Catholic University of Korea* 

Epistaxis occur due to trauma, disorders in mucosa or vessels, or coagulopathy. It is a very common disease, as 10% of all population experience severe epistaxis, about 30% of children aged 0~5, 56% of children aged 6~10 and 64% of children aged 11~15 are reported to

Although most cases of epistaxis are mild, that can be self-managed, life-threatening condition can be also possible. When encounter patient with severe epistaxis, it is important to find the bleeding focus and to analyse the causes of epistaxis fast and accurately, to treat the patient promptly to avoid complications such as hypotension, hypoxia, anemia,

Nasal bleeding can conveniently be divided into anterior and posterior epistaxis. Anterior bleeds come out the front of the nose, whereas posterior bleeds run down the back of the nose into the pharynx. Roughly 90% of cases of epistaxis can be classified as anterior. The common sites of anterior bleeding include the anterior aspect of the nasal septum, anterior edge of the inferior turbinate and the anterior ethmoid sinus. Among them, the anterior aspect of the nasal septum is the single most common site, where sometimes referred to as Kisselbach's plexus (Little's area). Kisselbach's plexus contains a rich capillary blood supply that is at the confluence of four different arterial blood supplies, which are sphenopalatine artery, greater palatine artery, superior labial artery, and anterior ethmoid artery (Figure 1). Posterior epistaxis typically arises from vessels on the posterior septum, on the floor of the nose in the posterior choana, or from the back of the middle or inferior turbinate. The area at the back of the inferior turbinate is specified as Woodruff's plexus (Figure 2). Recently, it is known that the Woodruff plexus is a venous plexus located at the back of the inferior

The causes of the epistaxis cannot be found in 80 to 90% of patients. It is easy to injure nasal mucosa and generate epistaxis, as it is rich with blood vessels just underneath mucosa. A number of factors and conditions contribute to the development, severity and recurrence of

experience one or more episode of epistaxis. (Cho, 2009 )

**1. Introduction** 

aspiration or death.

meatus, not an arterial plexus.

**3. Causes of epistaxis** 

epistaxis. (Table 1)

**2. Anatomy** 
