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Matrix recruitment and calcium sequestration for spatial specific otoconia development.

Yang H, Zhao X, Xu Y, Wang L, He Q, Lundberg YW - PLoS ONE (2011)

Bottom Line: In vivo, the presence of Oc90 in wildtype (wt) mice leads to an enrichment of Ca(2+) in the luminal matrices of the utricle and saccule, whereas absence of Oc90 in the mice leads to drastically reduced matrix-Ca(2+).Molecular modeling and sequence analysis predict structural features that may underlie the interaction and Ca(2+)-sequestering ability of these proteins.Together, the data provide a mechanism for the otoconial matrix assembly and the role of this matrix in accumulating micro-environmental Ca(2+) for efficient CaCO(3) crystallization, thus uncover a critical process governing spatial specific otoconia formation.

View Article: PubMed Central - PubMed

Affiliation: Vestibular Neurogenetics Laboratory, Boys Town National Research Hospital, Omaha, Nebraska, United States of America.

ABSTRACT
Otoconia are bio-crystals anchored to the macular sensory epithelium of the utricle and saccule in the inner ear for motion sensing and bodily balance. Otoconia dislocation, degeneration and ectopic calcification can have detrimental effects on balance and vertigo/dizziness, yet the mechanism underlying otoconia formation is not fully understood. In this study, we show that selected matrix components are recruited to form the crystal matrix and sequester Ca(2+) for spatial specific formation of otoconia. Specifically, otoconin-90 (Oc90) binds otolin through both domains (TH and C1q) of otolin, but full-length otolin shows the strongest interaction. These proteins have much higher expression levels in the utricle and saccule than other inner ear epithelial tissues in mice. In vivo, the presence of Oc90 in wildtype (wt) mice leads to an enrichment of Ca(2+) in the luminal matrices of the utricle and saccule, whereas absence of Oc90 in the mice leads to drastically reduced matrix-Ca(2+). In vitro, either Oc90 or otolin can increase the propensity of extracellular matrix to calcify in cell culture, and co-expression has a synergistic effect on calcification. Molecular modeling and sequence analysis predict structural features that may underlie the interaction and Ca(2+)-sequestering ability of these proteins. Together, the data provide a mechanism for the otoconial matrix assembly and the role of this matrix in accumulating micro-environmental Ca(2+) for efficient CaCO(3) crystallization, thus uncover a critical process governing spatial specific otoconia formation.

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Oc90 and otolin augment ECM calcification.Inorganic calcium deposits were visualized with Alizarin Red S staining, and averaged percentages of cells with ECM calcification over total cells were compared for different constructs and vectors. Under the same inducing conditions, untransfected cells and cells transfected with empty vectors (A) had similar ratios of calcification nodules, but both had significantly lower ratios than those transfected with otolin (B), Oc90 (C), or Oc90+otolin (D) (Figure 5B, 5C or 5D vs. 5A, p<0.001 as denoted by ***, n = 3 experiments x 3 fields for each). Figure 5E shows histograms of the averaged percentages of cells with ECM calcification. Co-transfection of Oc90 and otolin had a synergistic effect on calcification. # and ### indicate p<0.05 and 0.001, respectively, when pOc90 or pOc90+pOtolin is compared with pOtolin. $ indicates p<0.05 when pOc90+pOtolin is compared with pOc90.
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pone-0020498-g005: Oc90 and otolin augment ECM calcification.Inorganic calcium deposits were visualized with Alizarin Red S staining, and averaged percentages of cells with ECM calcification over total cells were compared for different constructs and vectors. Under the same inducing conditions, untransfected cells and cells transfected with empty vectors (A) had similar ratios of calcification nodules, but both had significantly lower ratios than those transfected with otolin (B), Oc90 (C), or Oc90+otolin (D) (Figure 5B, 5C or 5D vs. 5A, p<0.001 as denoted by ***, n = 3 experiments x 3 fields for each). Figure 5E shows histograms of the averaged percentages of cells with ECM calcification. Co-transfection of Oc90 and otolin had a synergistic effect on calcification. # and ### indicate p<0.05 and 0.001, respectively, when pOc90 or pOc90+pOtolin is compared with pOtolin. $ indicates p<0.05 when pOc90+pOtolin is compared with pOc90.

Mentions: We then established a cell culture system to test whether the presence of Oc90 and/or otolin can augment matrix calcification. NIH/3T3 cells stably transfected with Oc90 and/or otolin showed intense ECM calcification after 5 days of induction with 0.5 mM Ca2+ and 2 mM Pi (Figure 5). The Ca2+ concentration used here is much lower than that used in osteoblast calcification studies (a minimum of 2 mM for the latter); the Pi concentration is slightly lower too (a minimum of 2.5 mM for the latter) [42]. In the figure, inorganic calcium deposits were visualized with ARS staining, and percentages of cells with ECM calcification over total cells were obtained in each sample. No calcification was seen in untransfected or transfected cells cultured in standard media without additional supplements of Ca2+ and Pi (Figure S2). Under inducing conditions, ratios of cells with ECM calcification were similar between untransfected cells and cells transfected with empty vectors. Among cells stably transfected with otolin, an average of 4.0±0.4% had calcification nodules, as compared to 2.5±0.2% for those transfected with the empty vector under inducing conditions (Figure 5A & 5B) (p<0.001, n = 3 experiments x 3 survey fields in each). The effect of Oc90 on calcification (4.8±0.7%) was significantly stronger than that of otolin (p<0.05). Co-transfection of Oc90 and otolin had a synergistic effect on calcification, with 5.6±0.6% cells showing matrix calcification (p<0.05 vs. Oc90 transfectants, and p<0.001 vs. otolin transfectants). Due to the large numbers of total confluent cells in each view field, the percentages of cells with calcification nodules became small, but the differences between Oc90 (or otolin or Oc90+otolin) transfectants versus empty vectors were visually obvious even without a microscope. Therefore, the presence of Oc90 and/or otolin significantly increases the propensity of the extracellular matrix to calcify.


Matrix recruitment and calcium sequestration for spatial specific otoconia development.

Yang H, Zhao X, Xu Y, Wang L, He Q, Lundberg YW - PLoS ONE (2011)

Oc90 and otolin augment ECM calcification.Inorganic calcium deposits were visualized with Alizarin Red S staining, and averaged percentages of cells with ECM calcification over total cells were compared for different constructs and vectors. Under the same inducing conditions, untransfected cells and cells transfected with empty vectors (A) had similar ratios of calcification nodules, but both had significantly lower ratios than those transfected with otolin (B), Oc90 (C), or Oc90+otolin (D) (Figure 5B, 5C or 5D vs. 5A, p<0.001 as denoted by ***, n = 3 experiments x 3 fields for each). Figure 5E shows histograms of the averaged percentages of cells with ECM calcification. Co-transfection of Oc90 and otolin had a synergistic effect on calcification. # and ### indicate p<0.05 and 0.001, respectively, when pOc90 or pOc90+pOtolin is compared with pOtolin. $ indicates p<0.05 when pOc90+pOtolin is compared with pOc90.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105080&req=5

pone-0020498-g005: Oc90 and otolin augment ECM calcification.Inorganic calcium deposits were visualized with Alizarin Red S staining, and averaged percentages of cells with ECM calcification over total cells were compared for different constructs and vectors. Under the same inducing conditions, untransfected cells and cells transfected with empty vectors (A) had similar ratios of calcification nodules, but both had significantly lower ratios than those transfected with otolin (B), Oc90 (C), or Oc90+otolin (D) (Figure 5B, 5C or 5D vs. 5A, p<0.001 as denoted by ***, n = 3 experiments x 3 fields for each). Figure 5E shows histograms of the averaged percentages of cells with ECM calcification. Co-transfection of Oc90 and otolin had a synergistic effect on calcification. # and ### indicate p<0.05 and 0.001, respectively, when pOc90 or pOc90+pOtolin is compared with pOtolin. $ indicates p<0.05 when pOc90+pOtolin is compared with pOc90.
Mentions: We then established a cell culture system to test whether the presence of Oc90 and/or otolin can augment matrix calcification. NIH/3T3 cells stably transfected with Oc90 and/or otolin showed intense ECM calcification after 5 days of induction with 0.5 mM Ca2+ and 2 mM Pi (Figure 5). The Ca2+ concentration used here is much lower than that used in osteoblast calcification studies (a minimum of 2 mM for the latter); the Pi concentration is slightly lower too (a minimum of 2.5 mM for the latter) [42]. In the figure, inorganic calcium deposits were visualized with ARS staining, and percentages of cells with ECM calcification over total cells were obtained in each sample. No calcification was seen in untransfected or transfected cells cultured in standard media without additional supplements of Ca2+ and Pi (Figure S2). Under inducing conditions, ratios of cells with ECM calcification were similar between untransfected cells and cells transfected with empty vectors. Among cells stably transfected with otolin, an average of 4.0±0.4% had calcification nodules, as compared to 2.5±0.2% for those transfected with the empty vector under inducing conditions (Figure 5A & 5B) (p<0.001, n = 3 experiments x 3 survey fields in each). The effect of Oc90 on calcification (4.8±0.7%) was significantly stronger than that of otolin (p<0.05). Co-transfection of Oc90 and otolin had a synergistic effect on calcification, with 5.6±0.6% cells showing matrix calcification (p<0.05 vs. Oc90 transfectants, and p<0.001 vs. otolin transfectants). Due to the large numbers of total confluent cells in each view field, the percentages of cells with calcification nodules became small, but the differences between Oc90 (or otolin or Oc90+otolin) transfectants versus empty vectors were visually obvious even without a microscope. Therefore, the presence of Oc90 and/or otolin significantly increases the propensity of the extracellular matrix to calcify.

Bottom Line: In vivo, the presence of Oc90 in wildtype (wt) mice leads to an enrichment of Ca(2+) in the luminal matrices of the utricle and saccule, whereas absence of Oc90 in the mice leads to drastically reduced matrix-Ca(2+).Molecular modeling and sequence analysis predict structural features that may underlie the interaction and Ca(2+)-sequestering ability of these proteins.Together, the data provide a mechanism for the otoconial matrix assembly and the role of this matrix in accumulating micro-environmental Ca(2+) for efficient CaCO(3) crystallization, thus uncover a critical process governing spatial specific otoconia formation.

View Article: PubMed Central - PubMed

Affiliation: Vestibular Neurogenetics Laboratory, Boys Town National Research Hospital, Omaha, Nebraska, United States of America.

ABSTRACT
Otoconia are bio-crystals anchored to the macular sensory epithelium of the utricle and saccule in the inner ear for motion sensing and bodily balance. Otoconia dislocation, degeneration and ectopic calcification can have detrimental effects on balance and vertigo/dizziness, yet the mechanism underlying otoconia formation is not fully understood. In this study, we show that selected matrix components are recruited to form the crystal matrix and sequester Ca(2+) for spatial specific formation of otoconia. Specifically, otoconin-90 (Oc90) binds otolin through both domains (TH and C1q) of otolin, but full-length otolin shows the strongest interaction. These proteins have much higher expression levels in the utricle and saccule than other inner ear epithelial tissues in mice. In vivo, the presence of Oc90 in wildtype (wt) mice leads to an enrichment of Ca(2+) in the luminal matrices of the utricle and saccule, whereas absence of Oc90 in the mice leads to drastically reduced matrix-Ca(2+). In vitro, either Oc90 or otolin can increase the propensity of extracellular matrix to calcify in cell culture, and co-expression has a synergistic effect on calcification. Molecular modeling and sequence analysis predict structural features that may underlie the interaction and Ca(2+)-sequestering ability of these proteins. Together, the data provide a mechanism for the otoconial matrix assembly and the role of this matrix in accumulating micro-environmental Ca(2+) for efficient CaCO(3) crystallization, thus uncover a critical process governing spatial specific otoconia formation.

Show MeSH
Related in: MedlinePlus