**3. The roles of regulatory proteins in otoconia formation**

Otoconia formation depends on both organic and inorganic components that are secreted into the vestibular endolymph. Non-component regulatory proteins affect otoconia development and maintenance likely by several ways: (1) by influencing the secretion (Sollner et al. 2004), structural and functional modification of the component and anchoring proteins (Lundberg, unpublished data), and (2) by spatially and temporally increasing chemical gradients of Ca2+, HCO3-, H+ and possibly other ions/anions to establish an appropriate micro-environmental condition for crystal seeding and growth.

#### **3.1 NADPH oxidase 3 (Nox3) and associated proteins are essential for otoconia formation**

The Noxs are a family of enzymes whose primary function is to produce ROS (reactive oxygen species). These proteins participate in a wide range of pathological and physiological processes. To date, seven Nox family members, Nox1-Nox5, Duox1 and Duox2, have been identified in mammals (Bedard and Krause 2007). Noxs serve as the core catalytic components, and their activities are regulated by cytosolic partners such as p22*phox*, Nox organizers (Noxo1, p47phox and p40phox), and Nox activators (Noxa1 and p67phox).

Among the identified Nox family members, Nox3 is primarily expressed in the inner ear and is essential for otoconia development (Banfi et al. 2004; Cheng et al. 2001; Paffenholz et al. 2004). It interacts with p22*phox* and Noxo1 to form a functional NADPH oxidase complex, and all three components are required for otoconia development and normal balance in mice (Kiss et al. 2006; Nakano et al. 2007; Nakano et al. 2008; Paffenholz et al. 2004). However, the mechanisms underlying the requirement of Nox-related proteins for otoconia formation are poorly understood. One possible role of Nox3 is to oxidize otoconial proteins, including Oc90, which then undergo conformational changes to trigger crystal nucleation. Indeed, our recent unpublished data show that Nox3 modifies the structures of a few otoconia proteins (Xu et al. 2012).

A novel mechanism proposed by Nakano et al. (Nakano et al. 2008) states that while the Nox3 complex passes electrons from intracellular NADPH to extracellular oxygen, the plasma membrane becomes depolarized. Such depolarization of the apical membrane would elevate

Fetuin-A, also known as α2-HS-glycoprotein or countertrypin, is a hepatic secreted protein that promotes bone mineralization. It is among the most abundant non-collagen proteins found in bone (Quelch et al. 1984). Several recent studies demonstrated that fetuin-A can bind calcium and phosphate to form a calciprotein particle and prevent the precipitation of these minerals from serum (Heiss et al. 2003; Price et al. 2002), which may explain the role of fetuin-A in bone calcification and its potent inhibition of ectopic mineralization in soft tissues (Schafer et al. 2003; Westenfeld et al. 2007; Westenfeld et al. 2009). However, fetuin-A null mice have normal bone under regular dietary conditions (Jahnen-Dechent et al. 1997). Fetuin-A is present in otoconia crystals (Zhao et al. 2007), but null mice for the protein do not show balance deficits (Jahnen-Dechent, communication in Thalmann et al., 2006),

Taken together, findings on these low abundance otoconins indicate similarities and

Otoconia formation depends on both organic and inorganic components that are secreted into the vestibular endolymph. Non-component regulatory proteins affect otoconia development and maintenance likely by several ways: (1) by influencing the secretion (Sollner et al. 2004), structural and functional modification of the component and anchoring proteins (Lundberg, unpublished data), and (2) by spatially and temporally increasing chemical gradients of Ca2+, HCO3-, H+ and possibly other ions/anions to establish an

therefore, it is unlikely that the protein has a major impact on otoconia genesis.

differences between bone and otoconia biomineralization.

**formation** 

otoconia proteins (Xu et al. 2012).

**3. The roles of regulatory proteins in otoconia formation** 

appropriate micro-environmental condition for crystal seeding and growth.

**3.1 NADPH oxidase 3 (Nox3) and associated proteins are essential for otoconia** 

The Noxs are a family of enzymes whose primary function is to produce ROS (reactive oxygen species). These proteins participate in a wide range of pathological and physiological processes. To date, seven Nox family members, Nox1-Nox5, Duox1 and Duox2, have been identified in mammals (Bedard and Krause 2007). Noxs serve as the core catalytic components, and their activities are regulated by cytosolic partners such as p22*phox*, Nox organizers (Noxo1, p47phox and p40phox), and Nox activators (Noxa1 and p67phox).

Among the identified Nox family members, Nox3 is primarily expressed in the inner ear and is essential for otoconia development (Banfi et al. 2004; Cheng et al. 2001; Paffenholz et al. 2004). It interacts with p22*phox* and Noxo1 to form a functional NADPH oxidase complex, and all three components are required for otoconia development and normal balance in mice (Kiss et al. 2006; Nakano et al. 2007; Nakano et al. 2008; Paffenholz et al. 2004). However, the mechanisms underlying the requirement of Nox-related proteins for otoconia formation are poorly understood. One possible role of Nox3 is to oxidize otoconial proteins, including Oc90, which then undergo conformational changes to trigger crystal nucleation. Indeed, our recent unpublished data show that Nox3 modifies the structures of a few

A novel mechanism proposed by Nakano et al. (Nakano et al. 2008) states that while the Nox3 complex passes electrons from intracellular NADPH to extracellular oxygen, the plasma membrane becomes depolarized. Such depolarization of the apical membrane would elevate endolymphatic Ca2+ concentration by preventing cellular Ca2+ uptake from endolymph, and by increasing paracellular ion permeability to allow Ca2+ influx from perilymph to endolymph. In addition, Nox3-derived superoxide may react with endolymphatic protons and thereby elevate the pH so that CaCO3 can form and be maintained.
