ATP6V1H Deficiency Impairs Bone Development through Activation of MMP9 and MMP13.

ATP6V1H is a component of a large protein complex with vacuolar ATPase (V-ATPase) activity. We identified two generations of individuals in which short stature and osteoporosis co-segregated with a mutation in ATP6V1H. Since V-ATPases are highly conserved between human and zebrafish, we generated loss-of-function mutants in atp6v1h in zebrafish through CRISPR/Cas9-mediated gene knockout. Homozygous mutant atp6v1h zebrafish exhibited a severe reduction in the number of mature calcified bone cells and a dramatic increase in the expression of mmp9 and mmp13. Heterozygous adults showed curved vertebra that lack calcified centrum structure and reduced bone mass and density. Treatment of mutant embryos with small molecule inhibitors of MMP9 and MMP13 significantly restored bone mass in the atp6v1h mutants. These studies have uncovered a new, ATP6V1H-mediated pathway that regulates bone formation, and defines a new mechanism of disease that leads to bone loss. We propose that MMP9/MMP13 could be therapeutic targets for patients with this rare genetic disease

Correction: ATP6V1H Deficiency Impairs Bone Development through Activation of MMP9 and MMP13.

[This corrects the article DOI: 10.1371/journal.pgen.1006481.]

ATP6V1H Deficiency Impairs Bone Development through Activation of MMP9 and MMP13

<div><p>ATP6V1H is a component of a large protein complex with vacuolar ATPase (V-ATPase) activity. We identified two generations of individuals in which short stature and osteoporosis co-segregated with a mutation in <i>ATP6V1H</i>. Since V-ATPases are highly conserved between human and zebrafish, we generated loss-of-function mutants in <i>atp6v1h</i> in zebrafish through CRISPR/Cas9-mediated gene knockout. Homozygous mutant <i>atp6v1h</i> zebrafish exhibited a severe reduction in the number of mature calcified bone cells and a dramatic increase in the expression of <i>mmp9</i> and <i>mmp13</i>. Heterozygous adults showed curved vertebra that lack calcified centrum structure and reduced bone mass and density. Treatment of mutant embryos with small molecule inhibitors of MMP9 and MMP13 significantly restored bone mass in the <i>atp6v1h</i> mutants. These studies have uncovered a new, ATP6V1H-mediated pathway that regulates bone formation, and defines a new mechanism of disease that leads to bone loss. We propose that MMP9/MMP13 could be therapeutic targets for patients with this rare genetic disease.</p></div

Model of action.

<p>A schema showing Vacuolar ATPase and its subunits. Subunit H (V1, cytosolic domain) is shown in green. Under physiologic conditions, ATP hydrolysis occurs in the cytosolic domain while proton translocation occurs through the membrane domain. The V1H subunit activates ATP-driven proton pumping in the intact V-ATPase complex, but when V1 and V0 are disassociated, inhibition of the Mg-ATPase activity of the V1 domain occurs to prevent unnecessary ATP hydrolysis. When V1H is mutated, significantly higher levels of ATPase activity compared to the wild-type V1 complexes is expected, causing increased matrix degradation, leading to decreased bone density via increased MMP9/MMP13 activity.</p

Induction of <i>mmp9/mmp13</i> and rescue of atp6v1h-deficient phenotype.

<p>In (<b>A</b>), qPCR analysis of designated marker genes showing significant induction of <i>mmp9</i> and <i>mmp13</i> mRNA expression in <i>atp6v1h</i> mutant. Analysis of osteoclast cells by RNA whole mount <i>in situ</i> hybridization. <i>atp6v1h</i> mutant embryos showing significant increases of mmp9 and mmp13a signals in embryonic head skeleton (e/f/i/j wild type, g/h/k/l mutant), but osteoclast marker <i>rank</i> pattern is not changed (a/b wild type, c/d mutant) (<b>B</b>). In (<b>C</b>), analysis of <i>Mmp9/Mmp13</i> expression in mouse osteoclast Raw264.7 cells after knockdown of <i>Atp6v1h</i> by siRNA is shown. When <i>Atp6v1h</i> expression was knocked down by siRNA (a), the expression of <i>Mmp9</i> (b) and <i>Mmp13</i> (c) were induced. Similarly, the induction of <i>Mmp9</i> (d) and <i>Mmp13</i> (e) was also observed in RANKL-induced differentiated mouse osteoclast Raw264.7 cells. In (<b>D</b>), MMP inhibition rescues the bone phenotype in <i>Atp16V1h</i> mutant fish as detected by calcein staining; a, wild type, 0.5% DMSO; b, mutant, 0.5% DMSO; c, mutant treated with 200uM MMP9 inhibitor II in 0.5% DMSO; d, mutant treated with 20uM MMP13 inhibitor in 0.5% DMSO; e, mutant treated with 50uM MMP9/13 inhibitor I in 0.5% DMSO. Images are ventral views of 6 dpf embryos.</p

Micro-computed tomography scan of adult zebrafish.

<p>Adult zebrafish heterozygous for atp6V1h mutation (+/-) show curved body, compared to the wild type (+/+) sibling (<b>A</b>). X-ray of the same +/- fish shows curved spine compared to the +/+ sibling (<b>B</b>). Vertebra of +/- fish has bristlier surface and smaller size (<b>C</b>). Vertebra of +/- fish lacks calcified structure in the centrum cavity (<b>D</b>). Individual vertebra of +/- fish shows bristlier surface and smaller size with a hollow centrum (<b>E</b>) (n = 3 each).</p

Clinical features of patients with <i>ATP6V1H</i> mutations.

<p>The family comes from a three-generation pedigree; affected members are shaded in black (<b>A</b>); Chromatograms showing that the <i>ATP6V1H</i> mutation is present in affected family members, (II.1 and III.1 (<b>B</b>); DNA from I.1 is not available; (C) Radiographs (<b>C</b>) from Patient 1 (P1) (1, 2, and 3) and Patient 2 (P2) (4, and 5) reveal that P1 has scoliosis (C1) and increased radiolucency in tibia (C2). Bone scan shows multiple sites of increased bone mineral density uptake–tip of right scapula and multiple areas of thoracic and lumbar spine, sacrum, both sacroiliac joints, left humeral head, shaft of left femur, right tibia and right ribs (C3). Some increase in radiolucency especially in the lumbar spine (C4), is seen in P2.</p