Osteopontin is not Critical for Otoconia Formation or Balance Function

Abstract

Unlike the structural and mechanical role of bone crystals, the inertial mass of otoconia crystals provides a shearing force to stimulate the mechanoreceptors of the utricle and saccule (the gravity receptor organ) under the stimuli of linear motion. It is not clear whether otoconia, composed primarily of CaCO 3 and glycoproteins, go through similar calcification processes as bone. We have recently shown that otoconin-90 (Oc90) regulates the growth of otoconia crystals as osteopontin does bone crystals. Here, we analyzed the role of this non-collagenous bone matrix protein, osteopontin, in otoconia formation and balance function utilizing its knockout mice, whose inner ear phenotype has not been examined. Despite the presence of the protein in wild-type otoconia and vestibular hair cells, morphological, ultrastructural, and protein and calcium composition analyses of osteopontin null otoconia show that the protein is not needed for crystal formation, and no evidence of compensatory protein deposition is found. Employment of a wide spectrum of balance behavioral tests demonstrates that the protein is not critical for balance function either, which is confirmed by the normal function of the gravity receptor organ directly measured with linear vestibular-evoked potentials (VsEPs). When compared with findings on other otoconins, the data manifest a hierarchy of importance of proteins in crystallization and indicate mechanistic similarities and differences between bone and otoconia calcification.

Sparc Protein Is Required for Normal Growth of Zebrafish Otoliths

“…Because of the affinity of its N-terminal acidic module for both Ca 2+ and hydroxyapatite, Sparc may also act as a linker between collagen and the mineral phase in bone and promote mineralization. In vitro, however, Sparc has been variously found to stimulate (Termine et al 1981), inhibit (Romberg et al 1986Doi et al 1989), or not at all affect (Hunter et al 1996) (Tohse et al 2008), and osteopontin (Sakagami 2000;Thalmann et al 2006;Zhao et al 2008). Sparc-like itself, which is about twice the size of and 50-70% identical to Sparc, has been found in mouse otoconia .…”

“…Fourth, starmaker, a member of the secretory calcium-binding phosphoprotein family, in zebrafish (Söllner et al 2003) and otolith matrix macromolecule-64, its likely ortholog, in trout (Tohse et al 2008). Fifth, secreted phosphoprotein 1 or osteopontin, a secretory calcium-binding phosphoprotein prominent in bone and dentin, in mouse (Sakagami 2000;Thalmann et al 2006;Zhao et al 2008). Another half dozen proteins, including calbindin D-28K (Piscopo et al 2003(Piscopo et al , 2004, SPARC-like 1, countertrypin, and fetuin-A Zhao et al 2007), have been only located in otoconia but not yet characterized.…”

Proteins Involved in Otoconia Formation and Maintenance

“…Large otoconia but reduced in number (Legan et al 2000) Osteopontin Normal otoconia (Zhao et al 2008a) Fetuin-A Normal otoconia? (Xu et al 2010)…”

“…Osteopontin null mice have altered organization of bone matrix and weakened bone strength, leading to reduced bone fracture toughness (Duvall et al 2007;Thurner et al 2010). However, despite its presence in otoconia and vestibular sensory epithelia, osteopontin is dispensable for otoconia formation, and osteopontin knockout mice show normal vestibular morphology and balance function (Zhao et al 2008a). …”

“…The organic components of otoconia primarily consist of glycoproteins and proteoglycans Ito et al 1994;Pisam et al 2002;Pote and Ross 1991;Verpy et al 1999;Wang et al 1998;Xu et al 2010;Zhao et al, 2007). To date, as many as 8 murine otoconins have been identified (Table 1): the predominant otoconial protein, otoconin-90 (Oc90) and other 'minor' otoconins including otolin-1 (aka otolin) (Zhao et al 2007), fetuin-A (aka countertrypin) Zhao et al 2007), osteopontin (aka Spp1) (Sakagami 2000;Takemura et al 1994;Zhao et al 2008a), Sparc-like protein 1 (Sc1, aka hevin and Ecm2) Xu et al 2010), possibly secreted protein acidic and rich in cysteine (Sparc, aka BM-40 and osteonectin), and dentin matrix protein 1 (DMP1). Those otoconins are expressed in different cells and secreted into the utricular and saccular endolymph.…”